Inhibition of diacylglycerol kinase to augment adoptive t cell transfer

ABSTRACT

The present invention provides compositions and methods for inhibiting one or more diacylglycerol kinase (DGK) isoform in a cell in order to enhance the cytolytic activity of the cell. In one embodiment, the cells may be used in adoptive T cell transfer. For example, in some embodiments, the cell is modified to express a chimeric antigen receptor (CAR). Inhibition of DGK in T cells used in adoptive T cell transfer increases cytolytic activity of the T cells and thus may be used in the treatment of a variety of conditions, including cancer, infection, and immune disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is entitled to priority under 35 U.S.C. §119(e)to U.S. Provisional Patent Application 61/696,599, filed Sep. 4, 2012,the content of which is hereby incorporated by reference in its entiretyherein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under RO1AI058019 andP01 CA66726, awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Elicitation of T-cell effector responses requires signal transductionthrough the T-cell antigen receptor (TCR), a protein complex that bindsantigenic peptide presented by MHC, as well as through costimulatoryreceptors such as CD28. The effector responses generated from TCR signaltransduction differ across individual T-cell subsets that are classifiedaccording to the expression of cell surface molecules (Smith-Garvin etal., 2009, Annu Rev Immunol 27:591-619). Expression of the surfacemolecule CD8, for instance, identifies a subset of T cells that respondto antigenic peptides presented in the binding groove of MHC class I.CD8⁺ T cells are responsible for the recognition and elimination ofcells that express antigens derived from intracellular pathogens, suchas viruses and intracellular bacteria, and also mutated or embryonicproteins generated by cells that have undergone malignanttransformation. Although the extent to which CD8⁺ T cells are capable ofcontrolling the development and progression of tumorigenesis remainsuncertain, it is clear that deficiency of these cells increases thepotential for the development of malignancy and that enhanced functionof these CD8⁺ T cells can impart robust antitumor responses in bothanimal model systems and patients (Turcotte & Rosenberg, 2011, Adv Surg45:341-60; June, 2007, J Clin Invest 117:1466-76). It is also clear in anumber of models that although there may be an initial, potent CD8⁺T-cell response, this response is often insufficient to fully protectfrom tumors (Schreiber et al., 2011, Science 331:1565-70). Mechanismsunderlying this failure include (i) the lack of specific antigens withsufficient avidity for the TCR expressed by tumors, (ii) the absence ofcostimulatory ligands expressed by antigen-presenting cells (APC) withintumor-draining lymph nodes, and (iii) direct suppression of T-cellresponses within the tumor microenvironment mediated by inhibitorysecreted factors such as TGFβ, prostaglandin E (PGE)-2, or adenosine, aswell as inhibitory cells, such as regulatory T cells (Quezada et al.,2011, Immunol Rev 241:104-18).

The potential for effective responses by CD8⁺ T cells in some instancesof incurable malignancies, such as metastatic melanoma, has led tosignificant interest in defining ways to manipulate these cells togenerate more potent responses as well as responses against a morediverse array of tumors. One promising approach has focused onengineering T cells to express chimeric antigen receptors (CAR). CARsare transmembrane fusion proteins that consist of an extracellularantibody domain capable of binding to a specific tumor antigen coupledto intracellular signaling domains from TCR and costimulatory components(Milone et al., 2009, Mol Ther 17:1453-64). In principle, CARs provideseveral advantages over the endogenous receptors of T cells. First, theengineered ligand-binding segment of CARs arises from an antibody,obviating the need for MHC presentation. Second, the antibody-bindingcomponent of the CAR can be chosen to be both specific and highlysensitive to antigens expressed selectively by tumor cells, increasingavidity of the T cell-tumor interaction and minimizing the potential fordestruction of normal “bystander” host cells. Third, engagement of theCAR by ligand stimulates both TCR and costimulatory signaling modules,eliminating a requirement for expression of costimulatory ligands bytumor-draining APCs. CAR-expressing T cells that come into contact withtumor cells expressing the antigen of interest have been shown todevelop functional responses that lead to tumor cell lysis and cytokineproduction.

There has been considerable success in the use of CARs in animal models(Milone et al., 2009, Mol Ther 17:1453-64; Dower et al., 2000, NatImmunol 1:317-21), and recently, CAR-expressing T cells have been shownto be effective in patients to treat refractory chronic lymphocyticleukemias (CLL) (Chiang et al., 2007, J Clin Invest 117:1029-36; Loeseret al., 2007, J Exp Med 204:879-91). Although T cells engineered toexpress CARs are capable of overcoming some limitations of theendogenous immune system to combat tumors (e.g., CARs are not MHCrestricted and hence will lyse tumor cells that have downregulated MHCexpression), CAR-expressing T cells still lack intrinsic programming toovercome, perhaps, the most important component that limits CD8⁺ T-cellantitumor responses: the inhibitory tumor microenvironment.

However, there are a number of areas that appear to be importantpotential limitations to the success of CARs, especially for solidtumors. A major limitation is the loss of efficacy of the infused Tcells. As reported by others (Ahmadzadeh et al., 2009, Blood114:1537-1544; Whiteside, 2004, Cancer Immunol. Immunother. 53:865-878;Zippelius et al., 2004, Cancer Res. 64:2865-2873; Bronte et al., 2005,J. Exp. Med. 201:1257-1268; Prinz et al., 2012, J. Immunol.188:5990-6000; Monu et al., 2007, Cancer Res. 67:11447-11454; Janicki etal., 2008, Cancer Res. 68:2993-3000), even when the T cells successfullytraffic into tumors, in many instances, they appear to be inactivatedwithin tumors rather rapidly. The mechanisms are not fully understood,but probably involve secreted immune-inhibitory factors such as TGFβ,PGE2, and adenosine within the tumor microenvironment, interactions withinhibitory leukocytes (i.e. T-regulatory cells and myeloid suppressorcells) and/or contact with inhibitory molecules on the surface of tumorcells.

The mechanisms of T cell inactivation are not well understood,especially in CARs, where, unlike native T cells, the signaltransduction mechanisms have not been well studied. Thus, there is aneed in the art to develop compositions and methods for enhancing T cellactivation and killing ability during adoptive T cell transfer. Thepresent invention satisfies this unmet need.

SUMMARY OF THE INVENTION

The present invention provides a composition for enhancing the cytolyticactivity of a cell. In one embodiment, the composition comprises aninhibitor of diacylglycerol kinase (DGK) or a downstream effectorprotein thereof

In one embodiment, the inhibitor is selected from the group consistingof a small interfering RNA (siRNA), short hairpin RNA (shRNA), anantisense nucleic acid, a ribozyme, a dominant negative mutant, anantibody, a peptide, a zinc finger nuclease, and a small molecule.

In one embodiment, the cell is a T cell.

In one embodiment, the T cell is an activated T cell.

In one embodiment, the T cell is modified to express a chimeric antigenreceptor (CAR).

In one embodiment, the composition inhibits the DGK isoform selectedfrom the group consisting of DGKα and DGKζ.

In one embodiment, the composition inhibits both DGKα and DGKζ.

The invention provides an isolated cell having enhanced cytolyticactivity, wherein the cell comprises an inhibitor of DGK or a downstreameffector protein thereof.

The invention also provides a method of enhancing the cytolytic activityof a cell. In one embodiment, the method comprises administering to thecell an effective amount of a composition comprising an inhibitor of DGKor a downstream effector protein thereof.

In one embodiment, the inhibitor is selected from the group consistingof a small interfering RNA (siRNA), short hairpin RNA (shRNA), anantisense nucleic acid, a ribozyme, a dominant negative mutant, anantibody, a peptide, a zinc finger nuclease, and a small molecule.

In one embodiment, the cell is a T cell.

In one embodiment, the T cell is an activated T cell.

In one embodiment, the T cell is modified to express a chimeric antigenreceptor (CAR).

In one embodiment, the composition inhibits the DGK isoform selectedfrom the group consisting of DGKα and DGKζ.

In one embodiment, the composition inhibits both DGKα and DGKζ.

In one embodiment, the cell is genetically modified to express theinhibitor.

In one embodiment, administering the inhibitor comprises administeringthe inhibitor in an ex vivo environment.

The invention also provides a method of enhancing adoptive T celltransfer in a subject. In one embodiment, the method comprisesadministering to a T cell an effective amount of a compositioncomprising an inhibitor of DGK or a downstream effector protein thereof,wherein the T cell is administered to the subject during adoptive T celltransfer.

In one embodiment, the T cell is an activated T cell.

In one embodiment, the T cell is an autologous T cell. In oneembodiment, the T cell is modified to express a chimeric antigenreceptor (CAR).

In one embodiment, the composition inhibits the DGK isoform selectedfrom the group consisting of DGKα and DGKζ.

In one embodiment, the composition inhibits both DGKα and DGKζ.

In one embodiment, the cell is genetically modified to express theinhibitor.

In one embodiment, administering the inhibitor comprises administeringthe inhibitor in an ex vivo environment.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 is an illustration depicting a chimeric antigen receptor (CAR)construct.

FIG. 2 is a graph depicting the results from the transduction of mouse Tcells with retrovirus encoding a CAR. As depicted, transduction leads tosurface expression of the CAR on nearly 70% of T cells.

FIG. 3 is a graph depicting the results of an assay examining thepercent cell killing of AE17 tumor cells using mouse T cell CARs at avariety of ratios of T cell CARs per tumor cell.

FIG. 4 is a graph depicting the killing of tumor cells when using a 40:1ratio of T cells per one tumor cell.

FIG. 5 is a graph depicting the ability of the T cell CARs to kill tumorcells in wild-type, DGKα knockout, DGKζ knockout, or double DGKα/DGKζknockout T cells.

FIG. 6 is a graph depicting INFγ release by T cells induced by tumorcells.

FIG. 7 is a graph depicting the killing of tumor cells by wild type andDGK knockout meso-CAR T cells.

FIG. 8, comprising FIGS. 8A-8B, depicts how the elimination of DGKresulted in T cells less sensitive to TGFβ-mediated-inhibition of tumorkilling. FIG. 8A is a graph depicting how DGKα knockout cells areresistant to the inhibitory effects of TGFβ in a killing assay. FIG. 8Bis a graph depicting how DGKα knockout cells are resistant to theinhibitory effects of TGFβ in an INFγ release assay.

FIG. 9 is a graph depicting the effect of DGK knockout cells onpreventing tumor growth. The results of the WINN assay were collectedtwo weeks post-injection.

FIG. 10 is a graph depicting the effect of DGK knockout cells onpreventing tumor growth, when using an E:T ratio of 1:10.

FIG. 11 is a graph depicting the results of an intratumoral injectionstudy. The results comprise tumor measurements tabulated four dayspost-T cell injection.

FIG. 12 is a graph depicting the effect of DGK inhibitors on the killingactivity of human CAR-T cells (TIL) extracted from a tumor in an animalmodel.

FIG. 13 is an image demonstrating that various types of T cells wereinjected intratumorally and the size of tumors were measured after 4days. The DGKα knockout and DKO cells were more effective in reducingtumor size.

FIG. 14 is an image demonstrating that mice with established tumors wereinjected intravenously with 10⁷ wild type T cells or the three types ofDGK knockout T cells. Whereas the wild type meso-CAR T cells had no realantitumor effect, all three types of KO T cells reduced the tumor sizeby about 50%, showing they have much more anti-tumor activity than WTCAR T cells.

FIG. 15 is an image demonstrating the percent cell killing by tumorinfiltrating lymphocytes (TILs) in the presence and absence of DGKinhibitor.

FIG. 16, comprising FIG. 16A through FIG. 16D, depicts the results ofexperiments demonstrating that DGKζ-deficient activated CD8⁺ T cellsshow enhanced tumor responses in vivo. FIG. 16A: Twenty thousand naïve)(CD44^(lo)) CD8⁺ wild-type (wt) or DGKζ-deficient OT-I cells wereinjected intravenously into congenically marked (CD90.1) mice.Twenty-four hours later, the mice were injected intravenously with 5,000cfu Listeria-ova, and one week later, mice were euthanized, and thepresence of donor OT-I T cells (CD90.2⁺, ova tetramer+) was assessed(n=5, quantitation of 1 of 3 representative experiments is shown).CD90.2⁺ cells from FIG. 16A were isolated magnetically and 1×10⁶ cellswere injected intravenously into CD45.1⁺ mice bearing 2-week-oldsubcutaneous EL4-ova tumors. One week later, mice were euthanized andassessed for tumor size (FIG. 16B), persistence of donor (CD45.2⁺, ovatetramer+) T cells (FIG. 16C), and tumor-infiltrating donor T cells(FIG. 16D). “No T cell” mice did not receive donor T cells, and CD45.2cells were not detected in any organ tissue (FIG. 16B, data from 3pooled experiments. FIG. 16C and FIG. 16D, data from 1 of 3representative experiments, n=5 in each group).

FIG. 17, comprising FIG. 17A through FIG. 17C, depicts the results ofexperiments demonstrating enhanced CAR effector function inDGKζ-deficient CD8⁺ T cells. CD8⁺ wild-type (wt) or DGKζ-deficient OT-IT cells were isolated and transduced with mesoCAR retrovirus expressinggfp from an internal ribosomal entry site. FIG. 17A: Cells wereevaluated for expression of gfp, CD44 as a marker of activation, andovalbumin tetramer to assess TCR expression. B and C, mesoCAR-transducedwt or DGKζ-deficient OT-I cells were incubated with 5,000 AE17 ovalbumincells or AE17 cells expressing both mesothelin and ovalbumin at a ratioof 40:1 in a 96-well plate for 24 hours, and the presence of IFNγ (FIG.17B) or luciferase (cytotoxicity; FIG. 17C) in cell supernatants wasassessed. Calculated estimates of cytotoxicity were confirmed by visualevaluation of cell culture wells. One of the 3 representativeexperiments is shown; each well was carried out in triplicate. (FIG. 17Band FIG. 17C, P<0.0001 between DGK/AE17ova and wt/AE17ova orDGK/AE17ovameso and wt AE17ovameso. FIG. 17B, P=0.0327 betweenDGK/AE17ova and DGK/AE17ovameso, P=0.002 between wt/AE17ova andwt/AE17ovameso. FIG. 17 C, P<0.0001 between DGK/AE17ova andDGK/AE17ovameso, P=0.007 between wt/AE17ova and wt/AE17ovameso).

FIG. 18, comprising FIG. 18A through FIG. 18C, depicts the results ofexperiments demonstrating that deletion of both T-cell DGK isoformssignificantly enhances CAR-T cell effector functions. T cells wereisolated from wt, dgkα−/−, dgkζ−/−, or DKO mice, transduced withmesoCAR, and assessed for IFNγ production (FIG. 18A) and cytotoxicity oftarget cells (FIG. 18B and FIG. 18C) at indicated ratios as described inFIG. 17. In FIG. 18C, a ratio of 40:1 was used for experimental(AE17ovameso) or control (AE17ova) cell lines. One of the 3representative experiments is shown; each data point was conducted intriplicate (P for all mesoCAR-expressing constructs <0.0001 for AE17ovaand AE17ovameso).

FIG. 19, comprising FIG. 19A through FIG. 19C, depicts the results ofexperiments demonstrating enhanced CAR signaling in DGK-deficient CD8⁺ Tcells. FIG. 19A: A total of 1×10⁶ CAR-transduced (MesoCAR) or vectorcontrol (MIGR) CD8⁺ T cells were incubated with 4×10⁶ albumin-coatedbeads (alb) or mesothelin-coated beads (meso or M) or 2.5 mg/mL α-CD3(α-CD3ε or a) for the indicated times and assessed for phosphorylated(α-pERK) or total Erk (α-ERK) by immunoblotting. FIG. 19B: Levels ofpERK, total ERK, and actin were assessed after stimulation of 1×10⁶wild-type (wt) or dgkα−/−dgkζ−/− (double knockout; DKO) CD8⁺ CAR-T cellswith 4×10⁶ mesothelin-coated beads (M) or 2.5 mg/mL α-CD3 (α) for 15minutes. Each immunoblot analysis is a representative experiment from atleast 3 independent iterations. FIG. 19C: A total of 1×10⁶ wt orDKO-deficient CD8⁺ CAR T cells were incubated with 4×10⁶ beads coatedwith albumin (alb) or mesothelin (meso) for 5 hours and surfaceexpression of CD69 was assessed by flow cytometry.

FIG. 20, comprising FIG. 20A through FIG. 20C, depicts the results ofexperiments demonstrating that DGK-deficient mesoCAR-transduced T cellscontrol mesothelioma in vivo. FIG. 20A: A total of 1×10⁶ TClmeso cellswere coinjected subcutaneously with 2×10⁵ wild-type (wt)mesoCAR-transduced T cells or mesoCAR-transduced T cells lacking one orboth (DKO) of the indicated DGK. Ten days later, mice were euthanizedand tumors were measured. One of the 2 representative experiments(n=4-5) is shown. P of wt versus DKO mesoCAR T cells is 0.05. FIG. 20B:A total of 2×10⁶ AE17meso cells were injected into flanks of C57Bl/6mice. One week later, 1×10⁷ CAR-transduced T cells of indicated genotypewere injected intravenously into mice and tumors were measured atindicated time points (n=5 in each genotype, P for DKO CAR-T cellsversus wt CAR-T cells=0.0141 at day 10 posttransfer). FIG. 20C:Alternatively, mice were sacrificed 3 and 6 days after T-cell transferof indicated genotypes as in FIG. 20B and the presence of mesoCAR Tcells in tumor (top) or spleen (bottom) was determined [n=3 in eachgroup, 1 of 2 representative experiments. P=0.0082 (tumor) and 0.0461(spleen) at day 6; day 3 results did not differ significantly].

FIG. 21, comprising FIG. 21A and FIG. 21B, depicts the results ofexperiments demonstrating that DGK inhibitors enhance the cytotoxiccapacity of impaired human mesoCAR-transduced T cells. FIG. 21A:MesoCAR-transduced primary human cells were left unexposed or exposed toa human tumor line that does not express mesothelin (EM) or expresseshigh levels of mesothelin (EM-meso) for 96 hours. A total of 10⁵ T cellswere then isolated and recultured with 5×10³ luciferase-expressingEM-meso cells for 18 hours in the absence or presence of DGK inhibitorsR59022 (DGK1 Inhibitor) or R59949 (DGK2 inhibitor) and cell death oftarget cells was assessed by luciferase release (data from triplicatewells of one of the 3 representative experiments are shown. P of EM-mesoexposed T cells to EM exposed T cells=0.004 in no inhibitor group. P ofEM-meso-exposed T cells in the absence of inhibitor to DGK1inhibitor=0.006 or DGK2 inhibitor=0.003). FIG. 20B: Lysis of 5×10³EM-meso cells was assessed during incubation with no T cells or 10⁵untransduced or mesoCAR-transduced primary human T cell for 18 hours inthe presence or absence of the indicated concentration of TGFβ (datafrom triplicate wells of one of the 3 representative experiments. P ofmesoCAR T cells between 0 and 10 pg/mL=0.05, 0 and 100 pg/mL=0.025, and0 and 1000 pg/mL=0.017).

FIG. 22, comprising FIG. 22A and FIG. 22B, depicts the results ofexperiments demonstrating that DGK-deficient T cells are less inhibitedby TGFβ. A total of 1×10⁵ naïve (CD44^(lo)) mesoCAR-transduced T cellsof the indicated genotype were incubated with 5,000 AE17meso targetcells at a ratio of 40:1 in the absence or presence of TGFβ at theindicated concentration. IFNγ (FIG. 22A) and cytotoxicity (FIG. 22B) oftarget cells at 24 hours was assessed as described in FIG. 17. One ofthe 3 representative experiments is shown. Each data point was conductedin triplicate [P<0.0001 between wild-type (wt) and all DGK-deficient Tcells treated with TGFβ].

FIG. 23, comprising FIG. 23A and FIG. 23B, depicts the results ofexperiments demonstrating that MesoCAR-transduced DGKζ-deficient T cellsdemonstrate resistance to multiple inhibitory stimuli. MesoCARtransduced wt or DGKζ-deficient T cells were incubated with AE17mesotarget cells for 24 hours at a ratio of 40:1 in the presence or absenceof indicated concentrations of (FIG. 23A) adenosine or (FIG. 23B)prostaglandin E2 (PGE2) and cytotoxicity or IFNg production of targetcells was assessed as in FIG. 17.

FIG. 24, comprising FIG. 24A and FIG. 24B, depicts the results ofexperiments demonstrating that enhanced expression of FasL and TRAIL inDGK-deficient mesoCAR T cells. 2×10⁶ MesoCAR T cells replete (wt) for orlacking the indicated isoform of DGKs were stimulated with 2×10⁶mesothelin-coated beads for 18 hours in presence of IL2. FasL (FIG. 24A)and TRAIL (FIG. 24B) expression was determined by flow cytometry. One oftwo experimental iterations is shown, both experiments yielded similardata.

FIG. 25, comprising FIG. 25A and FIG. 25B, depicts the results ofexperiments demonstrating similar expression of Granzyme B and perforinin DGK-deficient mesoCAR T cells. Under conditions identical to FIG. 24,wt or DGK-deficient mesoCAR T cells were incubated withmesothelin-coated beads and the expression of perforin (FIG. 25A) andgranzyme B (FIG. 25B) was determined by flow cytometry using anintracellular staining protocol recommended by the manufacturer.

FIG. 26 is a schematic depicting the structure of FAP-CAR. Total RNA of73.3 hybridoma was extracted, reverse transcribed to cDNA, and PCRamplified and inserted into cloning vector to obtain the sequence ofvariable domains of IgG heavy and light chains. The anti-muFAP CARconsists of the anti-muFAP scFv, CD8α hinge and transmembrane domain,plus 4-1BB and CD3ζ intracellular signaling domains, and was cloned intoMigR1 retroviral vector in order to transduce primary mouse T cells.

FIG. 27 is a graph depicting the results of experiments demonstratingthat deletion of DGKζ enhanced cytolytic activity of FAP-CAR T cells.Splenic T cells were isolated from intact C57BL/6 mice, as well as DGKζknockout mice. Isolated T cells were then activated, transduced withFAP-CAR and expanded. A week later, FAP-CAR T cells with or without DGKζdeletion were reacted with 3T3 or 3T3.FAP fibroblasts for 18 hours todetermine cytotoxicity. * Denotes statistical significance betweenuntreated and two FAP-CAR-treated samples, p value <0.05. ^(#) Denotesstatistical significance between WT and DGKζ KO FAP-CAR-treated samples,p value <0.05.

FIG. 28 is a graph depicting the results of experiments demonstratingthat deletion of DGKζ enhanced IFNγ of FAP-CAR T cells. Splenic T cellswere isolated from intact C57BL/6 mice, as well as DGKζ knockout mice.Isolated T cells were then activated, transduced with FAP-CAR andexpanded. A week later, FAP-CAR T cells with or without DGKζ deletionwere reacted with 3T3 or 3T3.FAP fibroblasts for 18 hours to determineIFNγ production. * Denotes statistical significance between untreatedand two FAP-CAR-treated samples, p value <0.05. ^(#) Denotes statisticalsignificance between WT and DGKζ KO FAP-CAR-treated samples, p value<0.05.

FIG. 29 is a graph depicting the results of experiments demonstratingthat DGKζ knockout FAP-T cells persisted longer than wildtype FAP-CAR Tcells. AE17.ova tumor mice were adoptively transferred with 10 millionwildtype or DGKζ knockout FAP-CAR T cells when tumors reached 100 mm³Tumors were harvested 11 days post-injection to determine persistence ofT cells. Percent FAP-CAR T cells were determined using flow cytometry. *Denotes statistical significance between WT and DGKζ KO FAP-CAR-treatedsamples, p value <0.05.

FIG. 30 is a graph depicting the results of experiments demonstrating anenhanced therapeutic response of FAP-CAR T cells conferred by deletionof a negative T cell regulator DGKζ. Mice with AE17.ova flank tumorswere injected intravenously with FAP-CAR T cells with or withoutdeletion of DGKζ when tumors were approximately 75 mm³ The tumormeasurements were then followed.* Denotes statistical significancebetween untreated and FAP-CAR-treated samples, p value <0.05. ^(#)Denotes statistical significance between single dose FAP-CAR treatedgroup versus DGKζ KO FAP-CAR treated group

DETAILED DESCRIPTION

The present invention provides compositions and methods for regulatingDiacylglycerol Kinase (DGK) in a cell, preferably a T cell. Theinvention is based upon the discovery that blockade of DGK isoformsenhances the cytolytic activity and persistence of T cells. In oneembodiment, the invention provides inhibition of one or more DGK isoformin a T cell in order to augment adoptive T cell transfer.

Accordingly, the present invention provides compositions and methods forinhibiting one or more DGK isoform for augmenting cytolytic activity ina cell. In one embodiment, inhibiting one or more DGK isoform enhancescytolytic activity in T cell modified to express a desired protein(e.g., a chimeric antigen receptor (CAR)). That is, the invention isbased on the discovery that inhibition of DGK enhances tumor killingactivity of CAR modified T cells. However, the invention should not belimited to only T cells expressing a CAR. Rather, the invention includesany T cell, genetically modified or not, expressing a gene of interestfor adoptive T cell transfer.

In one embodiment, the present invention provides methods for treating awide variety of conditions by inhibiting one or more DGK isoform duringadoptive T cell transfer. In one embodiment, T cells are geneticallymodified ex vivo to express at least one inhibitor. The present methodsare useful for treating any condition where adoptive T cell transfer isused. Exemplary conditions for which the present method can be used totreat include various types of cancers, HIV, Hepatitis C, immunedisorders, bacterial infections, viral infections, and the like.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

“Activation”, as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. An antibody of theinvention includes intracellularly expressed antibody, or intrabody.Antibodies can be intact immunoglobulins derived from natural sources orfrom recombinant sources and can be immunoreactive portions of intactimmunoglobulins. Antibodies are typically tetramers of immunoglobulinmolecules. The antibodies in the present invention may exist in avariety of forms including, for example, polyclonal antibodies,monoclonal antibodies, Fv, Fab and F(ab)₂, as well as single chainantibodies, human antibodies, and humanized antibodies (Harlow et al.,1999, In: Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY; Harlow et al., 1989, In: Antibodies: A LaboratoryManual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)₂, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. κ and λ, light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

“Antisense” refers particularly to the nucleic acid sequence of thenon-coding strand of a double stranded DNA molecule encoding apolypeptide, or to a sequence which is substantially homologous to thenon-coding strand. As defined herein, an antisense sequence iscomplementary to the sequence of a double stranded DNA molecule encodinga polypeptide. It is not necessary that the antisense sequence becomplementary solely to the coding portion of the coding strand of theDNA molecule. The antisense sequence may be complementary to regulatorysequences specified on the coding strand of a DNA molecule encoding apolypeptide, which regulatory sequences control expression of the codingsequences.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is mistakenly recognized by the immune system asbeing foreign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia greata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, is reduced.

An “effective amount” or “therapeutically effective amount” of acompound is that amount of compound which is sufficient to provide abeneficial effect to the subject to which the compound is administered.An “effective amount” of a delivery vehicle is that amount sufficient toeffectively bind or deliver a compound.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “heterologous” as used herein is defined as DNA or RNAsequences or proteins that are derived from the different species.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared X 100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a compound, composition, vector,or delivery system of the invention in the kit for effecting alleviationof the various diseases or disorders recited herein. Optionally, oralternately, the instructional material can describe one or more methodsof alleviating the diseases or disorders in a cell or a tissue of amammal. The instructional material of the kit of the invention can, forexample, be affixed to a container which contains the identifiedcompound, composition, vector, or delivery system of the invention or beshipped together with a container which contains the identifiedcompound, composition, vector, or delivery system. Alternatively, theinstructional material can be shipped separately from the container withthe intention that the instructional material and the compound be usedcooperatively by the recipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, i.e., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, i.e., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, i.e., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (i.e.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of the tumorantigen is intended to indicate an abnormal level of expression of thetumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). Examples of subjectsinclude humans, dogs, cats, mice, rats, and transgenic species thereof.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology, for the purpose of diminishing oreliminating those signs.

As used herein, “treating a disease or disorder” means reducing thefrequency with which a symptom of the disease or disorder is experiencedby a patient. Disease and disorder are used interchangeably herein.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention provides compositions and methods for modulatingDiacylgylceral Kinase (DGK) activity in a cell, preferably a T cell. Theinvention also provides compounds and methods of modulating downstreamtargets of DGK and its functional equivalents. In one embodiment, theinvention is used to augment adoptive T cell transfer in the treatmentof a wide variety of conditions, including, but not limited to cancer,infections, and immune disorders.

The present invention is based on the discovery that removal or blockadeof DGK isoforms enhances the efficacy of T cells used in adoptive T celltransfer. In certain embodiments, the T cell is an activated T cell. Inone embodiment, the T cells used in adoptive T cell transfer aremodified to express a desired protein (e.g., a chimeric antigen receptor(CAR)). However, the invention should not be limited to only T cellsexpressing a CAR. Rather, the invention includes any T cell, geneticallymodified or not, expressing a gene of interest for adoptive T celltransfer.

In one embodiment, T cells are genetically modified ex vivo to expressan inhibitor of one or more DGK isoforms. An advantage of a geneticapproach to DGK inhibition is that the effects will be isolated only tothe transfused T cells, thus minimizing systemic toxicity and enhancingsafety. For example, T cells can be modified to by introducing specificinhibitors of DGK including but not limited to, a siRNA, shRNA, amicroRNA, an antisense nucleic acid, a ribozyme, a dominant negativemutant, an intracellular antibody, a peptide, a zinc finger nuclease,and a small molecule. It is described herein, that expression of such aninhibitor will augment tumor killing efficacy.

The present invention provides methods for treating a variety ofconditions including, but not limited to, cancer, infection, and immunedisorders. In one embodiment, inhibition of one or more DGK isoformsenhances T cell cytolytic activity, thereby reducing the size and growthof a tumor. In one embodiment, the methods comprise inhibiting one ormore DGK isoform in a T cell directed to target a specific antigen. Inone embodiment, the methods comprise inhibiting one or more DGK isoformin a T cell genetically modified to express a CAR. In one embodiment,the CAR directs the modified T cell to a specific tumor antigen. Themethods of the present invention augment CAR T cell therapy by enhancingthe cytolytic activity of the CAR modified T cells.

In one embodiment, inhibiting DGK in a T cell results in T cells thatare less capable of “turning-off” DAG-mediated signaling within T cellswhereby the DGK inhibited T cells are less sensitive to inhibition oftumor killing mediated by inhibitor signals in the tumormicroenvironment. For example, the present invention is partly basedupon the discovery that PGE2, adenosine, and TGFβ were less able tosuppress effector functions in T cells that lacked one or both T-cellisoforms of DGK. The resistance to the inhibitory effects of PGE2,adenosine, and TGFβ is an important factor in a therapeutic contextsince tumors make large amounts of these inhibitors of tumor killing.

Compositions

The invention is based partly on the discovery that inhibition of DGKactivity or expression improves the cytolytic activity of T cells, forexample T cells genetically modified to express a chimeric antigenreceptor (CAR). However, the invention should not be limited to only Tcells expressing a CAR. Rather, the invention includes any T cell,genetically modified or not, expressing a gene of interest for adoptiveT cell transfer. As such, the data presented herein indicates thattherapeutic inhibition of DGK and/or its down-stream effector proteinsmay be beneficial, in addition to other effects, by providing sustainedtargeted lysis of unwanted cells (i.e. tumor cells).

The present invention relates to the discovery that inhibition of anyone or more DGK isoform in a cell, preferably a T cell, provides atherapeutic benefit. Thus, the invention comprises compositions andmethods for modulating any of these proteins in a T cell therebyenhancing cytolytic activity of the T cell.

The present invention includes a generic concept for inhibiting one ormore DGK isoform or any component of the signal transduction pathwayassociated with the phosphorylation of diacyglycerol (DAG), therebyincreasing the cytolytic activity of the T cell. In one embodiment, thecomposition of the invention inhibits DGKα. In one embodiment, thecomposition of the invention inhibits DGKζ.

In one embodiment, the invention comprises a composition for enhancingthe cytolytic activity of a T cell. In certain embodiments, the T cellis an activated T cell. In one embodiment, the T cell is a geneticallymodified T cell. In one embodiment the T cell is modified to express aCAR. The composition comprises an inhibitor of any one or more DGKisoforms. In one embodiment, the DGK inhibitor of the invention includesany one or more compositions disclosed in U.S. Patent ApplicationPublication No. 20050266510, the entire content of which is incorporatedherein by reference. In another embodiment, the composition comprisingthe DGK inhibitor is selected from the group consisting of a smallinterfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA, anantisense nucleic acid, a ribozyme, a dominant negative mutant, anintracellular antibody, a peptide, a zinc finger nuclease, and a smallmolecule. However, the invention should not be limited these types ofinhibitors. Rather, any type of inhibitor known in the art or to beidentified can be used to inhibit DGK. For example, techniques recentlypublished to knock down RNA or delete genes include Transcriptionactivator-like effector nucleases (TALENs) and CRISPR technology(Pennisi, 2013 Science 341: 833).

In one embodiment, the DGK inhibitor of the invention is an interferingRNA which reduces translation of at least one DGK isoform. Aninterfering RNA can include a siRNA, a shRNA, and a microRNA. An siRNApolynucleotide is an RNA nucleic acid molecule that interferes with RNAactivity that is generally considered to occur via apost-transcriptional gene silencing mechanism. An siRNA polynucleotidepreferably comprises a double-stranded RNA (dsRNA) but is not intendedto be so limited and may comprise a single-stranded RNA (see, e.g.,Martinez et al., 2002 Cell 110:563-74). The siRNA polynucleotideincluded in the invention may comprise other naturally occurring,recombinant, or synthetic single-stranded or double-stranded polymers ofnucleotides (ribonucleotides or deoxyribonucleotides or a combination ofboth) and/or nucleotide analogues as provided herein (e.g., anoligonucleotide or polynucleotide or the like, typically in 5′ to 3′phosphodiester linkage). Accordingly it will be appreciated that certainexemplary sequences disclosed herein as DNA sequences capable ofdirecting the transcription of the siRNA polynucleotides are alsointended to describe the corresponding RNA sequences and theircomplements, given the well-established principles of complementarynucleotide base-pairing.

Preferred siRNA polynucleotides comprise double-stranded polynucleotidesof about 18-30 nucleotide base pairs, preferably about 18, about 19,about 20, about 21, about 22, about 23, about 24, about 25, about 26, orabout 27 base pairs, and in other preferred embodiments about 19, about20, about 21, about 22 or about 23 base pairs, or about 27 base pairs,whereby the use of “about” indicates that in certain embodiments andunder certain conditions the processive cleavage steps that may giverise to functional siRNA polynucleotides that are capable of interferingwith expression of a selected polypeptide may not be absolutelyefficient. Hence, siRNA polynucleotides, may include one or more siRNApolynucleotide molecules that may differ (e.g., by nucleotide insertionor deletion) in length by one, two, three, four or more base pairs as aconsequence of the variability in processing, in biosynthesis, or inartificial synthesis of the siRNA. The siRNA polynucleotide of thepresent invention may also comprise a polynucleotide sequence thatexhibits variability by differing (e.g., by nucleotide substitution,including transition or transversion) at one, two, three or fournucleotides from a particular sequence. These differences can occur atany of the nucleotide positions of a particular siRNA polynucleotidesequence, depending on the length of the molecule, whether situated in asense or in an antisense strand of the double-stranded polynucleotide.The nucleotide difference may be found on one strand of adouble-stranded polynucleotide, where the complementary nucleotide withwhich the substitute nucleotide would typically form hydrogen bond basepairing, may not necessarily be correspondingly substituted. Inpreferred embodiments, the siRNA polynucleotides are homogeneous withrespect to a specific nucleotide sequence.

Based on the present disclosure, it should be appreciated that thesiRNAs of the present invention may effect silencing of the targetpolypeptide expression to different degrees. The siRNAs thus must firstbe tested for their effectiveness. Selection of siRNAs are madetherefrom based on the ability of a given siRNA to interfere with ormodulate the expression of the target polypeptide. Accordingly,identification of specific siRNA polynucleotide sequences that arecapable of interfering with expression of a desired target polypeptiderequires production and testing of each siRNA. The methods for testingeach siRNA and selection of suitable siRNAs for use in the presentinvention are fully set forth herein the Examples. Since not all siRNAsthat interfere with protein expression will have a physiologicallyimportant effect, the present disclosure also sets forth variousphysiologically relevant assays for determining whether the levels ofinterference with target protein expression using the siRNAs of theinvention have clinically relevant significance.

It is appreciated by one skilled in the art, that siRNAs are easilydesigned and manufactured. Further, effects of siRNA are typicallytransient in nature, which make them optimal for certain therapies wheresustained inhibition is undesired. Another form of an interfering RNA,shRNA polynucleotides utilize the endogenous processing machinery of thecell and are often designed for high potency, sustainable effects, andfewer off-target effects (Rao et al., 2009, Adv Drug Deliv Rev, 61:746-759). As would be understood by those skilled in the art, thepresent invention encompasses both siRNA and shRNA polynucleotides,which can be designed and delivered to inhibit one or more DGK isoform.

One skilled in the art will readily appreciate that as a result of thedegeneracy of the genetic code, many different nucleotide sequences mayencode the same polypeptide. That is, an amino acid may be encoded byone of several different codons, and a person skilled in the art canreadily determine that while one particular nucleotide sequence maydiffer from another, the polynucleotides may in fact encode polypeptideswith identical amino acid sequences. As such, polynucleotides that varydue to differences in codon usage are specifically contemplated by thepresent invention.

One skilled in the art will appreciate, based on the disclosure providedherein, that one way to decrease the mRNA and/or protein levels of oneor more DGK isoform in a cell is by reducing or inhibiting expression ofthe nucleic acid encoding the DGK isoform. Thus, the protein level ofthe DGK isoform in a cell can also be decreased using a molecule orcompound that inhibits or reduces gene expression such as, for example,an antisense molecule or a ribozyme.

In a preferred embodiment, the modulating sequence is an antisensenucleic acid sequence which is expressed by a plasmid vector. Theantisense expressing vector is used to transfect a mammalian cell or themammal itself, thereby causing reduced endogenous expression of adesired protein in the cell. However, the invention should not beconstrued to be limited to inhibiting expression of a protein bytransfection of cells with antisense molecules. Rather, the inventionencompasses other methods known in the art for inhibiting expression oractivity of a protein in the cell including, but not limited to, the useof a ribozyme, the expression of a non-functional protein (i.e. dominantnegative mutant) and use of an intracellular antibody.

Antisense molecules and their use for inhibiting gene expression arewell known in the art (see, e.g., Cohen, 1989, In:Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRCPress). Antisense nucleic acids are DNA or RNA molecules that arecomplementary, as that term is defined elsewhere herein, to at least aportion of a specific mRNA molecule (Weintraub, 1990, ScientificAmerican 262:40). In the cell, antisense nucleic acids hybridize to thecorresponding mRNA, forming a double-stranded molecule therebyinhibiting the translation of genes.

The use of antisense methods to inhibit the translation of genes isknown in the art, and is described, for example, in Marcus-Sakura (1988,Anal. Biochem. 172:289). Such antisense molecules may be provided to thecell via genetic expression using DNA encoding the antisense molecule astaught by Inoue, 1993, U.S. Pat. No. 5,190,931.

Alternatively, antisense molecules of the invention may be madesynthetically and then provided to the cell. Antisense oligomers ofbetween about 10 to about 30, and more preferably about 15 nucleotides,are preferred, since they are easily synthesized and introduced into atarget cell. Synthetic antisense molecules contemplated by the inventioninclude oligonucleotide derivatives known in the art which have improvedbiological activity compared to unmodified oligonucleotides (see U.S.Pat. No. 5,023,243).

Ribozymes and their use for inhibiting gene expression are also wellknown in the art (see, e.g., Cech et al., 1992, J. Biol. Chem.267:17479-17482; Hampel et al., 1989, Biochemistry 28:4929-4933;Eckstein et al., International Publication No. WO 92/07065; Altman etal., U.S. Pat. No. 5,168,053). Ribozymes are RNA molecules possessingthe ability to specifically cleave other single-stranded RNA in a manneranalogous to DNA restriction endonucleases. Through the modification ofnucleotide sequences encoding these RNAs, molecules can be engineered torecognize specific nucleotide sequences in an RNA molecule and cleave it(Cech, 1988, J. Amer. Med. Assn. 260:3030). A major advantage of thisapproach is the fact that ribozymes are sequence-specific.

There are two basic types of ribozymes, namely, tetrahymena-type(Hasselhoff, 1988, Nature 334:585) and hammerhead-type. Tetrahymena-typeribozymes recognize sequences which are four bases in length, whilehammerhead-type ribozymes recognize base sequences 11-18 bases inlength. The longer the sequence, the greater the likelihood that thesequence will occur exclusively in the target mRNA species.Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating specific mRNA species, and18-base recognition sequences are preferable to shorter recognitionsequences which may occur randomly within various unrelated mRNAmolecules.

Ribozymes useful for inhibiting the expression of a protein may bedesigned by incorporating target sequences into the basic ribozymestructure which are complementary to the mRNA sequence of the desiredprotein of the present invention, including but not limited to one ormore DGK isoform, and equivalents thereof. Ribozymes targeting thedesired protein may be synthesized using commercially available reagents(Applied Biosystems, Inc., Foster City, Calif.) or they may begenetically expressed from DNA encoding them.

In another aspect of the invention, the protein can be inhibited by wayof inactivating and/or sequestering the DGK protein. As such, inhibitingthe effects of a protein can be accomplished by using a dominantnegative mutant. Alternatively an antibody specific for the desiredprotein, otherwise known as an antagonist to the protein may be used. Inone embodiment, the antagonist is a protein and/or compound having thedesirable property of interacting with a binding partner of the proteinand thereby competing with the corresponding wild-type protein. Inanother embodiment, the antagonist is a protein and/or compound havingthe desirable property of interacting with the protein and therebysequestering the protein.

In another aspect of the invention, expression of the protein isinhibited by a zinc finger nuclease administered to a cell. In general,a zinc finger nuclease comprises a zinc finger binding domain and acleavage domain.

Zinc finger binding domains may be engineered to recognize and bind toany nucleic acid sequence of choice, for example a nucleic acid sequenceassociated with the expression of one or more DGK isforms. See, forexample, Beerli et al. (2002) Nat. Biotechnol. 20:135-141; Pabo et al.(2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nat.Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol.12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416;Zhang et al. (2000) J. Biol. Chem. 275 (43):33850-33860; Doyon et al.(2008) Nat. Biotechnol. 26:702-708; and Santiago et al. (2008) Proc.Natl. Acad. Sci. USA 105:5809-5814. An engineered zinc finger bindingdomain may have a novel binding specificity compared to anaturally-occurring zinc finger protein. Engineering methods include,but are not limited to, rational design and various types of selection.Rational design includes, for example, using databases comprisingdoublet, triplet, and/or quadruplet nucleotide sequences and individualzinc finger amino acid sequences, in which each doublet, triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and6,534,261, the disclosures of which are incorporated by reference hereinin their entireties. As an example, the algorithm of described in U.S.Pat. No. 6,453,242 may be used to design a zinc finger binding domain totarget a preselected sequence.

A zinc finger binding domain may be designed to recognize a DNA sequenceranging from about 3 nucleotides to about 21 nucleotides in length, orfrom about 8 to about 19 nucleotides in length. In general, the zincfinger binding domains of the zinc finger nucleases disclosed hereincomprise at least three zinc finger recognition regions (i.e., zincfingers). In one embodiment, the zinc finger binding domain may comprisefour zinc finger recognition regions. In another embodiment, the zincfinger binding domain may comprise five zinc finger recognition regions.In still another embodiment, the zinc finger binding domain may comprisesix zinc finger recognition regions. A zinc finger binding domain may bedesigned to bind to any suitable target DNA sequence. See for example,U.S. Pat. Nos. 6,607,882; 6,534,261 and 6,453,242, the disclosures ofwhich are incorporated by reference herein in their entireties.

Exemplary methods of selecting a zinc finger recognition region mayinclude phage display and two-hybrid systems, and are disclosed in U.S.Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248;6,140,466; 6,200,759; and 6,242,568; as well as WO 98/37186; WO98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237, each of which isincorporated by reference herein in its entirety. In addition,enhancement of binding specificity for zinc finger binding domains hasbeen described, for example, in WO 02/077227.

In some embodiments, the zinc finger nuclease may further comprise anuclear localization signal or sequence (NLS). A NLS is an amino acidsequence which facilitates targeting the zinc finger nuclease proteininto the nucleus to introduce a double stranded break at the targetsequence in the chromosome. Nuclear localization signals are known inthe art. See, for example, Makkerh et al. (1996) Current Biology6:1025-1027.

A zinc finger nuclease also includes a cleavage domain. The cleavagedomain portion of the zinc finger nucleases disclosed herein may beobtained from any endonuclease or exonuclease. Non-limiting examples ofendonucleases from which a cleavage domain may be derived include, butare not limited to, restriction endonucleases and homing endonucleases.See, for example, 2002-2003 Catalog, New England Biolabs, Beverly,Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388 orwww.neb.com. Additional enzymes that cleave DNA are known (e.g., 51Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease;yeast HO endonuclease). See also Linn et al. (eds.) Nucleases, ColdSpring Harbor Laboratory Press, 1993. One or more of these enzymes (orfunctional fragments thereof) may be used as a source of cleavagedomains.

A cleavage domain also may be derived from an enzyme or portion thereof,as described above, that requires dimerization for cleavage activity.Two zinc finger nucleases may be required for cleavage, as each nucleasecomprises a monomer of the active enzyme dimer. Alternatively, a singlezinc finger nuclease may comprise both monomers to create an activeenzyme dimer. As used herein, an “active enzyme dimer” is an enzymedimer capable of cleaving a nucleic acid molecule. The two cleavagemonomers may be derived from the same endonuclease (or functionalfragments thereof), or each monomer may be derived from a differentendonuclease (or functional fragments thereof).

When two cleavage monomers are used to form an active enzyme dimer, therecognition sites for the two zinc finger nucleases are preferablydisposed such that binding of the two zinc finger nucleases to theirrespective recognition sites places the cleavage monomers in a spatialorientation to each other that allows the cleavage monomers to form anactive enzyme dimer, e.g., by dimerizing. As a result, the near edges ofthe recognition sites may be separated by about 5 to about 18nucleotides. For instance, the near edges may be separated by about 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides. It willhowever be understood that any integral number of nucleotides ornucleotide pairs may intervene between two recognition sites (e.g., fromabout 2 to about 50 nucleotide pairs or more). The near edges of therecognition sites of the zinc finger nucleases, such as for examplethose described in detail herein, may be separated by 6 nucleotides. Ingeneral, the site of cleavage lies between the recognition sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31, 978-31, 982. Thus, a zinc finger nucleasemay comprise the cleavage domain from at least one Type IIS restrictionenzyme and one or more zinc finger binding domains, which may or may notbe engineered. Exemplary Type IIS restriction enzymes are described forexample in International Publication WO 07/014,275, the disclosure ofwhich is incorporated by reference herein in its entirety. Additionalrestriction enzymes also contain separable binding and cleavage domains,and these also are contemplated by the present disclosure. See, forexample, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

A zinc finger nuclease may be engineered to introduce a double strandedbreak at the targeted site of integration. The double stranded break maybe at the targeted site of integration, or it may be up to 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or 1000 nucleotides awayfrom the site of integration. In some embodiments, the double strandedbreak may be up to 1, 2, 3, 4, 5, 10, 15, or 20 nucleotides away fromthe site of integration. In other embodiments, the double stranded breakmay be up to 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides away fromthe site of integration. In yet other embodiments, the double strandedbreak may be up to 50, 100, or 1000 nucleotides away from the site ofintegration.

Modification of Nucleic Acid Molecules

Inhibition of one or more DGK isoform can be accomplished using anucleic acid molecule. For example, the inhibitor is selected from thegroup consisting of a small interfering RNA (siRNA), short hairpin RNA(shRNA), a microRNA, an antisense nucleic acid, a ribozyme, a zincfinger nuclease, an expression vector encoding a dominant negativemutant, and the likes.

By way of example, modification of nucleic acid molecules is describedin the context of an siRNA molecule. However, the methods of modifyingnucleic acid molecules can be applied to other types of nucleic acidbased inhibitors of the invention.

Polynucleotides of the siRNA may be prepared using any of a variety oftechniques, which are useful for the preparation of specifically desiredsiRNA polynucleotides. For example, a polynucleotide may be amplifiedfrom a cDNA prepared from a suitable cell or tissue type. Such apolynucleotide may be amplified via polymerase chain reaction (PCR).Using this approach, sequence-specific primers are designed based on thesequences provided herein, and may be purchased or synthesized directly.An amplified portion of the primer may be used to isolate a full-lengthgene, or a desired portion thereof, from a suitable DNA library usingwell known techniques. A library (cDNA or genomic) is screened using oneor more polynucleotide probes or primers suitable for amplification.Preferably, the library is size-selected to include largerpolynucleotide sequences. Random primed libraries may also be preferredin order to identify 5′ and other upstream regions of the genes. Genomiclibraries are preferred for obtaining introns and extending 5′sequences. The siRNA polynucleotide contemplated by the presentinvention may also be selected from a library of siRNA polynucleotidesequences.

For hybridization techniques, a partial polynucleotide sequence may belabeled (e.g., by nick-translation or end-labeling with ³²P) using wellknown techniques. A bacterial or bacteriophage library may then bescreened by hybridization to filters containing denatured bacterialcolonies (or lawns containing phage plaques) with the labeled probe(see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 2001).Hybridizing colonies or plaques are selected and expanded, and the DNAis isolated for further analysis.

Alternatively, numerous amplification techniques are known in the artfor obtaining a full-length coding sequence from a partial cDNAsequence. Within such techniques, amplification is generally performedvia PCR. One such technique is known as “rapid amplification of cDNAends” or RACE (see, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y., 2001).

A number of specific siRNA polynucleotide sequences useful forinterfering with target polypeptide expression are presented in theExamples, the Drawings, and in the Sequence Listing included herein.siRNA polynucleotides may generally be prepared by any method known inthe art, including, for example, solid phase chemical synthesis.Modifications in a polynucleotide sequence may also be introduced usingstandard mutagenesis techniques, such as oligonucleotide-directedsite-specific mutagenesis. Further, siRNAs may be chemically modified orconjugated with other molecules to improve their stability and/ordelivery properties. Included as one aspect of the invention are siRNAsas described herein, wherein one or more ribose sugars has been removedtherefrom.

Alternatively, siRNA polynucleotide molecules may be generated by invitro or in vivo transcription of suitable DNA sequences (e.g.,polynucleotide sequences encoding a target polypeptide, or a desiredportion thereof), provided that the DNA is incorporated into a vectorwith a suitable RNA polymerase promoter (such as for example, T7, U6,H1, or SP6 although other promoters may be equally useful). In addition,an siRNA polynucleotide may be administered to a mammal, as may be a DNAsequence (e.g., a recombinant nucleic acid construct as provided herein)that supports transcription (and optionally appropriate processingsteps) such that a desired siRNA is generated in vivo.

In one embodiment, an siRNA polynucleotide, wherein the siRNApolynucleotide is capable of interfering with expression of a targetpolypeptide can be used to generate a silenced cell. Any siRNApolynucleotide that, when contacted with a biological source for aperiod of time, results in a significant decrease in the expression ofthe target polypeptide is included in the invention. Preferably thedecrease is greater than about 10%, more preferably greater than about20%, more preferably greater than about 30%, more preferably greaterthan about 40%, about 50%, about 60%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95% or about 98% relative to the expressionlevel of the target polypeptide detected in the absence of the siRNA.Preferably, the presence of the siRNA polynucleotide in a cell does notresult in or cause any undesired toxic effects, for example, apoptosisor death of a cell in which apoptosis is not a desired effect of RNAinterference.

In another embodiment, the siRNA polynucleotide that, when contactedwith a biological source for a period of time, results in a significantdecrease in the expression of the target polypeptide. Preferably thedecrease is about 10%-20%, more preferably about 20%-30%, morepreferably about 30%-40%, more preferably about 40%-50%, more preferablyabout 50%-60%, more preferably about 60%-70%, more preferably about70%-80%, more preferably about 80%-90%, more preferably about 90%-95%,more preferably about 95%-98% relative to the expression level of thetarget polypeptide detected in the absence of the siRNA. Preferably, thepresence of the siRNA polynucleotide in a cell does not result in orcause any undesired toxic effects.

In yet another embodiment, the siRNA polynucleotide that, when contactedwith a biological source for a period of time, results in a significantdecrease in the expression of the target polypeptide. Preferably thedecrease is about 10% or more, more preferably about 20% or more, morepreferably about 30% or more, more preferably about 40% or more, morepreferably about 50% or more, more preferably about 60% or more, morepreferably about 70% or more, more preferably about 80% or more, morepreferably about 90% or more, more preferably about 95% or more, morepreferably about 98% or more relative to the expression level of thetarget polypeptide detected in the absence of the siRNA. Preferably, thepresence of the siRNA polynucleotide in a cell does not result in orcause any undesired toxic effects.

Any polynucleotide of the invention may be further modified to increaseits stability in vivo. Possible modifications include, but are notlimited to, the addition of flanking sequences at the 5′ and/or 3′ ends;the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterlinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine, and wybutosine and the like, as well asacetyl- methyl-, thio- and other modified forms of adenine, cytidine,guanine, thymine, and uridine.

Antibodies

The concept of “Intracellular Immunization” or “IntracellularInhibition” has in the last decade emerged as an important strategy tocounteract functionalities of pathogenic bacteria, viruses andparasites. Intracellular Immunization utilizes molecular modulators suchas anti-sense RNA, ribozymes, dominant negative mutants andintracellular antibodies (intrabodies) for inhibiting functional geneexpression within the cell. Previous studies have shown the efficacy ofintrabodies (e.g., sFvs and Fabs) targeting expression in differentcompartments of the cell, including the nucleus, ER, cytoplasm, golgi,plasma membrane, mitochondria, where they act to counteract antigens ormolecules in a specific pathway. (Marasco, W. A., et al, Proc. Natl.Acad. Sci., USA 90:7889-7893 (1993); Chen, S. Y., et al., Human GeneTherapy 5:595-601 (1994); Chen, S. Y., et al., Proc Natl Acad Sci, USA91:5932-5936 (1994); Mhashilkar, A. M., et al., Embo J 14:1542-1551(1995); Marasco, W. A., et al. Gene Therapy 4:11-15 (1997); Richardson,J. H., et al., Proc Natl Acad Sci, USA 92:3137-3141 (1995); Duan, L., etal., Human Gene Therapy 5:1315-1324 (1994)). The antibodies can belocalized to specific cellular compartments, e.g., the ER, nucleus,inner surface of the plasma membrane, the cytoplasm and themitochondria. (See e.g., Marasco et al, 1993; Mhashilkar et al., 1995;Biocca et al., 1995).

As will be understood by one skilled in the art, any antibody that canrecognize and bind to an antigen of interest is useful in the presentinvention. Preferably, the antibody is an intracellularly expressedantibody, or otherwise referred to as an intrabody. That is, theantibody can inhibit one or more DGK isoforms to provide a beneficialeffect, in addition to other effects, by enhancing cytolytic activity ofa T cell.

Accordingly, the invention provides a single domain intrabody that bindsto an intracellular (cytosolic) protein or intracellular domain of aprotein (e.g., one or more DGK isoforms). The intrabody is used to,e.g., specifically inhibit an enzymatic activity of the intracellularprotein or domain. Intracellular proteins and domains that can betargets for the intrabody include kinases, a proteases, nucleases,telomerases, transferases, reductases, hydrolyases, and isomerases. Inan embodiment of the invention, the intrabody is specific for one ormore DGK isoforms.

The intrabody comprises whole antibodies, heavy chains, Fab′ fragments,single-chain antibodies and diabodies. In one preferred method of thepresent invention, the intrabody comprises a single-chain antibody(sFv). The antibodies for use in the present invention can be obtainedby methods known in the art against the antigen of interest. Forexample, single chain antibodies are prepared according to the teachingof PCT/US93/06735, filed on Jan. 17, 1992 and U.S. patent applicationSer. No. 08/350,215, filed on Dec. 6, 1994, incorporated herein byreference.

According to the invention, an intrabody is created that specificallyinhibits an intracellular protein or protein activity. The intrabody canbe selected to bind to an intracellular protein of any organism.Notably, because of the degree of evolutionary conservation of enzymes,protein-protein interactions, and signal transduction pathways, eventhough the intrabody will preferably be specific for an enzyme in acell, it can also be expected that the intrabody will inhibit ahomologous enzyme in a related species.

Domain intrabodies specific for any particular enzyme or catalyticregion thereof can be readily identified by screening a single domainantibody library. Antibody engineering has enabled the production ofsingle domain antibody libraries, and such libraries have beenconstructed from a number of variable domain scaffolds, including humanV_(H) or Y_(L) (Jespers, L. et al., 2004, J. Mol. Biol. 337:893-903),camelid V_(H) (Tanha, J. et al., 2001, J. Biol. Chem. 276:24774-80), andshark V-NAR (Nuttall, S. D., et al., 2004, Proteins 55:187-97).Libraries from other species exist as well. However, to avoid adverseimmune responses when domain intrabodies are administered to a subject,it is generally preferable that source of domain intrabody correspond tothe subject to which the intrabody will be administered.

In an embodiment of the invention, domain intrabodies are obtained byselecting a single variable domain from a variable region of an antibodyhaving two variable domains (i.e., a heterodimer of a heavy chainvariable domain and a light chain variable domain). Methods forobtaining heavy chain-light chain heterodimers include, for example, theimmunological method described by Kohler and Milstein, Nature256:495-497 (1975) and Campbell, Monoclonal Antibody Technology, TheProduction and Characterization of Rodent and Human Hybridomas, Burdonet al., Eds., Laboratory Techniques in Biochemistry and MolecularBiology, Volume 13, Elsevier Science Publishers, Amsterdam (1985); aswell as by the recombinant. DNA methods such as described by Huse et al,Science 246, 1275-81. (1989). The antibodies can also be obtained fromphage display or yeast surface display libraries bearing combinations ofV_(H) and V_(L) domains in the form of scFv or Fab. The V_(H) and V_(L)domains can be encoded by nucleotides that are synthetic, partiallysynthetic, or naturally derived. Single variable domain antibodies canalso be found in Fab and scFv phage display libraries (Cai, X. et al.,1996, Proc. Natl. Acad. Sci. USA. 93:6280-5). In certain embodiments,phage display libraries bearing human antibody fragments are preferred.Other sources of human antibodies are transgenic mice engineered toexpress human immunoglobulin genes.

The invention also includes functional equivalents of the antibodiesdescribed herein. Functional equivalents have binding characteristicscomparable to those of the antibodies, and include, for example,hybridized and single chain antibodies, as well as fragments thereof.Methods of producing such functional equivalents are disclosed in PCTApplication WO 93/21319 and PCT Application WO 89/09622.

Functional equivalents include polypeptides with amino acid sequencessubstantially the same as the amino acid sequence of the variable orhypervariable regions of the antibodies. “Substantially the same” aminoacid sequence is defined herein as a sequence with at least 70%,preferably at least about 80%, more preferably at least about 90%, evenmore preferably at least about 95%, and most preferably at least 99%homology to another amino acid sequence (or any integer in between 70and 99), as determined by the FASTA search method in accordance withPearson and Lipman, 1988 Proc. Nat'l. Acad. Sci. USA 85: 2444-2448Chimeric or other hybrid antibodies have constant regions derivedsubstantially or exclusively from human antibody constant regions andvariable regions derived substantially or exclusively from the sequenceof the variable region of a monoclonal antibody from each stablehybridoma.

Genetic Modification

The invention includes an isolated nucleic acid operably linked to anucleic acid comprising a promoter/regulatory sequence such that thenucleic acid is preferably capable of directing expression of theprotein encoded by the nucleic acid. Thus, the invention encompassesexpression vectors and methods for the introduction of exogenous DNAinto cells with concomitant expression of the exogenous DNA in the cellssuch as those described, for example, in Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York), and in Ausubel et al. (1997, Current Protocols in MolecularBiology, John Wiley & Sons, New York).

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity.

In one embodiment, the expression of natural or synthetic nucleic acidsencoding the inhibitor is typically achieved by operably linking anucleic acid to a promoter, and incorporating the construct into anexpression vector. The vectors can be suitable for replication andintegration eukaryotes. Typical cloning vectors contain transcriptionand translation terminators, initiation sequences, and promoters usefulfor regulation of the expression of the desired nucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector.

The desired nucleic can be cloned into a number of types of vectors.However, the present invention should not be construed to be limited toany particular vector. Instead, the present invention should beconstrued to encompass a wide plethora of vectors which are readilyavailable and/or well-known in the art. For example, a desiredpolynucleotide of the invention can be cloned into a vector including,but not limited to a plasmid, a phagemid, a phage derivative, an animalvirus, and a cosmid. Vectors of particular interest include expressionvectors, replication vectors, probe generation vectors, and sequencingvectors.

In specific embodiments, the expression vector is selected from thegroup consisting of a viral vector, a bacterial vector and a mammaliancell vector. Numerous expression vector systems exist that comprise atleast a part or all of the compositions discussed above. Prokaryote-and/or eukaryote-vector based systems can be employed for use with thepresent invention to produce polynucleotides, or their cognatepolypeptides. Many such systems are commercially and widely available.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001), and in Ausubel et al.(1997), and in other virology and molecular biology manuals. Viruses,which are useful as vectors include, but are not limited to,retroviruses, adenoviruses, adeno-associated viruses, herpes viruses,and lentiviruses. In general, a suitable vector contains an origin ofreplication functional in at least one organism, a promoter sequence,convenient restriction endonuclease sites, and one or more selectablemarkers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

For expression of the desired polynucleotide, at least one module ineach promoter functions to position the start site for RNA synthesis.The best known example of this is the TATA box, but in some promoterslacking a TATA box, such as the promoter for the mammalian terminaldeoxynucleotidyl transferase gene and the promoter for the SV40 genes, adiscrete element overlying the start site itself helps to fix the placeof initiation.

Additional promoter elements, i.e., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either co-operativelyor independently to activate transcription.

A promoter may be one naturally associated with a gene or polynucleotidesequence, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment and/or exon. Such a promoter canbe referred to as “endogenous.” Similarly, an enhancer may be onenaturally associated with a polynucleotide sequence, located eitherdownstream or upstream of that sequence. Alternatively, certainadvantages will be gained by positioning the coding polynucleotidesegment under the control of a recombinant or heterologous promoter,which refers to a promoter that is not normally associated with apolynucleotide sequence in its natural environment. A recombinant orheterologous enhancer refers also to an enhancer not normally associatedwith a polynucleotide sequence in its natural environment. Suchpromoters or enhancers may include promoters or enhancers of othergenes, and promoters or enhancers isolated from any other prokaryotic,viral, or eukaryotic cell, and promoters or enhancers not “naturallyoccurring,” i.e., containing different elements of differenttranscriptional regulatory regions, and/or mutations that alterexpression. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (U.S. Pat. No.4,683,202, U.S. Pat. No. 5,928,906). Furthermore, it is contemplated thecontrol sequences that direct transcription and/or expression ofsequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the celltype, organelle, and organism chosen for expression. Those of skill inthe art of molecular biology generally know how to use promoters,enhancers, and cell type combinations for protein expression, forexample, see Sambrook et al. (2001). The promoters employed may beconstitutive, tissue-specific, inducible, and/or useful under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.However, other constitutive promoter sequences may also be used,including, but not limited to the simian virus 40 (SV40) early promoter,mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV)long terminal repeat (LTR) promoter, Moloney virus promoter, the avianleukemia virus promoter, Epstein-Barr virus immediate early promoter,Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the muscle creatine promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter in the invention provides a molecularswitch capable of turning on expression of the polynucleotide sequencewhich it is operatively linked when such expression is desired, orturning off the expression when expression is not desired. Examples ofinducible promoters include, but are not limited to a metallothioninepromoter, a glucocorticoid promoter, a progesterone promoter, and atetracycline promoter. Further, the invention includes the use of atissue specific promoter, which promoter is active only in a desiredtissue. Tissue specific promoters are well known in the art and include,but are not limited to, the HER-2 promoter and the PSA associatedpromoter sequences.

In order to assess the expression of the inhibitor, the expressionvector to be introduced into a cell can also contain either a selectablemarker gene or a reporter gene or both to facilitate identification andselection of expressing cells from the population of cells sought to betransfected or infected through viral vectors. In other embodiments, theselectable marker may be carried on a separate piece of DNA and used ina co-transfection procedure. Both selectable markers and reporter genesmay be flanked with appropriate regulatory sequences to enableexpression in the host cells. Useful selectable markers are known in theart and include, for example, antibiotic-resistance genes, such as neoand the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Reportergenes that encode for easily assayable proteins are well known in theart. In general, a reporter gene is a gene that is not present in orexpressed by the recipient organism or tissue and that encodes a proteinwhose expression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells.

Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (see, e.g.,Ui-Tei et al., 2000 FEBS Lett. 479:79-82). Suitable expression systemsare well known and may be prepared using well known techniques orobtained commercially. Internal deletion constructs may be generatedusing unique internal restriction sites or by partial digestion ofnon-unique restriction sites. Constructs may then be transfected intocells that display high levels of siRNA polynucleotide and/orpolypeptide expression. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast or insectcell by any method in the art. For example, the expression vector can betransferred into a host cell by physical, chemical or biological means.It is readily understood that the introduction of the expression vectorcomprising the polynucleotide of the invention yields a silenced cellwith respect to a protein.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York), and in Ausubel et al. (1997, Current Protocols in MolecularBiology, John Wiley & Sons, New York).

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Apreferred colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (i.e., an artificial membrane vesicle). Thepreparation and use of such systems is well known in the art.

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

In one embodiment, the inhibitor of the invention encompasses in vitrotranscribed RNA. In one embodiment, an in vitro transcribed RNA can beintroduced to a cell as a form of transient transfection. The RNA isproduced by in vitro transcription using a polymerase chain reaction(PCR)-generated template. DNA of interest from any source can bedirectly converted by PCR into a template for in vitro mRNA synthesisusing appropriate primers and RNA polymerase. The source of the DNA canbe, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, syntheticDNA sequence or any other appropriate source of DNA.

In one embodiment, the DNA to be used for PCR contains an open readingframe. The DNA can be from a naturally occurring DNA sequence from thegenome of an organism. In one embodiment, the DNA is a full length geneof interest of a portion of a gene. The gene can include some or all ofthe 5′ and/or 3′ untranslated regions (UTRs). The gene can include exonsand introns. In one embodiment, the DNA to be used for PCR is a humangene. In another embodiment, the DNA to be used for PCR is a human geneincluding the 5′ and 3′ UTRs. The DNA can alternatively be an artificialDNA sequence that is not normally expressed in a naturally occurringorganism. An exemplary artificial DNA sequence is one that containsportions of genes that are ligated together to form an open readingframe that encodes a fusion protein. The portions of DNA that areligated together can be from a single organism or from more than oneorganism.

Genes that can be used as sources of DNA for PCR include genes thatencode polypeptides that provide a therapeutic or prophylactic effect toan organism or that can be used to diagnose a disease or disorder in anorganism. Preferred genes are genes which are useful for a short termtreatment, or where there are safety concerns regarding dosage or theexpressed gene. For example, for treatment of cancer, autoimmunedisorders, parasitic, viral, bacterial, fungal or other infections, thetransgene(s) to be expressed may encode a polypeptide that functions asa ligand or receptor for cells of the immune system, or can function tostimulate or inhibit the immune system of an organism. In someembodiments, t is not desirable to have prolonged ongoing stimulation ofthe immune system, nor necessary to produce changes which last aftersuccessful treatment, since this may then elicit a new problem. Fortreatment of an autoimmune disorder, it may be desirable to inhibit orsuppress the immune system during a flare-up, but not long term, whichcould result in the patient becoming overly sensitive to an infection.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. “Substantially complementary”, as used herein,refers to sequences of nucleotides where a majority or all of the basesin the primer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a genethat is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a gene that encodes a particular domain of interest. In oneembodiment, the primers are designed to amplify the coding region of ahuman cDNA, including all or portions of the 5′ and 3′ UTRs. Primersuseful for PCR are generated by synthetic methods that are well known inthe art. “Forward primers” are primers that contain a region ofnucleotides that are substantially complementary to nucleotides on theDNA template that are upstream of the DNA sequence that is to beamplified. “Upstream” is used herein to refer to a location 5, to theDNA sequence to be amplified relative to the coding strand. “Reverseprimers” are primers that contain a region of nucleotides that aresubstantially complementary to a double-stranded DNA template that aredownstream of the DNA sequence that is to be amplified. “Downstream” isused herein to refer to a location 3′ to the DNA sequence to beamplified relative to the coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA preferably has 5′ and3′ UTRs. In one embodiment, the 5′ UTR is between zero and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one preferred embodiment, the promoter isa T7 polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In a preferred embodiment, the mRNA has both a cap on the 5′ end and a3′ poly(A) tail which determine ribosome binding, initiation oftranslation and stability mRNA in the cell. On a circular DNA template,for instance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA.

5′ caps on also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun, 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Modified Cell

In one embodiment, the instant invention provides a cell-based systemfor expressing an inhibitor that is capable of inhibiting one or moreDGK isoform. The invention includes a cell that has been modified topossess heightened cytolytic activity as compared to an otherwiseidentical cell. The modified cell is suitable for administration to amammalian recipient alone or in combination with other therapies.

In one embodiment, the instant invention provides a cell for use inadoptive T cell transfer, wherein the T cell is modified to express aninhibitor that is capable of inhibiting one or more DGK isoform. In oneembodiment, the invention provides a T cell modified to express a CARand an inhibitor capable for inhibiting one or more DGK isoform.

In general, a CAR and CAR modified T cells are described inPCT/US2011/064191, which is incorporated herein by reference. In oneembodiment, a CAR comprises an extracellular domain and an intracellulardomain. The extracellular domain comprises a target-specific bindingelement otherwise referred to as an antigen binding moiety. Theintracellular domain or otherwise the cytoplasmic domain comprises, acostimulatory signaling region and a zeta chain portion. Thecostimulatory signaling region refers to a portion of the CAR comprisingthe intracellular domain of a costimulatory molecule. Costimulatorymolecules are cell surface molecules other than antigens receptors ortheir ligands that are required for an efficient response of lymphocytesto antigen.

As would be understood by those skilled in the art, CAR modified T cellscan be generated by any method known in the art. For example, the CARmodified T cells can be generated by introducing an expression vectorencoding the CAR to a T cell, as described elsewhere herein. In oneembodiment, the DGK inhibited cells of the invention are generated by amethod comprising administering an expression vector which encodes a CARand further encodes the inhibitor of the invention, to a T cell. Inanother embodiment, the method comprises administering an expressionvector encoding a CAR and an expression vector encoding the inhibitor,to the T cell.

In another embodiment, the CAR modified T cells can be generated byadministering in vitro transcribed RNA encoding a CAR, as describedelsewhere herein. In one embodiment, the DGK inhibited cells of theinvention are generated by a method comprising transfecting an RNA whichencodes a CAR and further encodes the inhibitor of the invention, to a Tcell. In another embodiment, the method comprises transfecting an RNAencoding a CAR and an RNA encoding the inhibitor, to the T cell.

In another embodiment, the DGK inhibited cells of the invention aregenerated by a method comprising transfecting an expression vectorencoding a CAR and an RNA encoding the inhibitor, to a T cell. In yetanother embodiment, the DGK inhibited cells of the invention aregenerated by a method comprising transfecting an RNA encoding a CAR andan expression vector encoding the inhibitor, to a T cell.

In some embodiments, the nucleic acids of the invention are deliveredinto cells using a retroviral or lentiviral vector. Retroviral andlentiviral vectors can be delivered into different types of eukaryoticcells as well as into tissues and whole organisms using transduced cellsas carriers or cell-free local or systemic delivery of encapsulated,bound or naked vectors. The method used can be for any purpose wherestable expression is required or sufficient.

In other embodiments, the nucleic acids of the invention are deliveredinto cells using in vitro transcribed mRNA. In vitro transcribed mRNAcan be delivered into different types of eukaryotic cells as well asinto tissues and whole organisms using transfected cells as carriers orcell-free local or systemic delivery of encapsulated, bound or nakedmRNA. The method used can be for any purpose where transient expressionis required or sufficient.

The disclosed methods can be applied to the modulation of T cellactivity in basic research and therapy, in the fields of cancer, stemcells, acute and chronic infections, and autoimmune diseases, includingthe assessment of the ability of the genetically modified T cell to killa target cancer cell.

Sources of T Cells

Prior to expansion and genetic modification of the T cells of theinvention, a source of T cells is obtained from a subject. T cells canbe obtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors. In certain embodiments of the presentinvention, any number of T cell lines available in the art, may be used.In certain embodiments of the present invention, T cells can be obtainedfrom a unit of blood collected from a subject using any number oftechniques known to the skilled artisan, such as Ficoll™ separation. Inone preferred embodiment, cells from the circulating blood of anindividual are obtained by apheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets. In one embodiment, the cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In oneembodiment of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Again, surprisingly, initial activation steps in theabsence of calcium lead to magnified activation. As those of ordinaryskill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other salinesolution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyresuspended in culture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺T cells, canbe further isolated by positive or negative selection techniques. Forexample, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmune-compromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainembodiments, it may be desirable to perform the selection procedure anduse the “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁺ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62L^(hi), GITR⁺, and FoxP3⁺. Alternatively, in certainembodiments, T regulatory cells are depleted by anti-C25 conjugatedbeads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8⁺ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4⁺ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8⁺ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

Whether prior to or after genetic modification of the T cells to expressa desirable protein (e.g., a CAR), the T cells can be activated andexpanded generally using methods as described, for example, in U.S. Pat.Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent ApplicationPublication No. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For costimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In one embodiment, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4⁺ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe invention, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular embodiment, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1particles per T cell. In one embodiment, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle: cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer,preferably PBS (without divalent cations such as, calcium andmagnesium). Again, those of ordinary skill in the art can readilyappreciate any cell concentration may be used. For example, the targetcell may be very rare in the sample and comprise only 0.01% of thesample or the entire sample (i.e., 100%) may comprise the target cell ofinterest. Accordingly, any cell number is within the context of thepresent invention. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in one embodiment, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain in certain embodiments. For example, using highconcentration of cells allows more efficient selection of CD8+ T cellsthat normally have weaker CD28 expression.

In one embodiment of the present invention, the mixture may be culturedfor several hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment of the invention the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

Therapeutic Application

The present invention includes an inhibitor of any one or more DGKisoform in a cell. The invention also includes a cell having heightedcytolytic activity wherein one or more DGK isoform in the cell has beeninhibited.

In one embodiment, the DGK inhibitor of the invention can beadministered to a subject in need thereof. When the DGK inhibitor of theinvention is prepared for administration, it is preferably combined witha pharmaceutically acceptable carrier, diluent or excipient to form apharmaceutical formulation, or unit dosage form. The total activeingredients in such formulations include from 0.1 to 99.9% by weight ofthe formulation. A “pharmaceutically acceptable” is a carrier, diluent,excipient, and/or salt that is compatible with the other ingredients ofthe formulation, and not deleterious to the recipient thereof. Theactive ingredient for administration may be present as a powder or asgranules; as a solution, a suspension or an emulsion.

Pharmaceutical formulations containing the DGK inhibitors of theinvention can be prepared by procedures known in the art using wellknown and readily available ingredients. The DGK inhibitor of theinvention can also be formulated as solutions appropriate for parenteraladministration, for instance by intramuscular, subcutaneous orintravenous routes.

In one embodiment, the present invention includes a type of cellulartherapy where T cells are genetically modified to express a DGKinhibitor and the modified T cell is infused to a recipient in needthereof. In one embodiment, the modified T cell is modified to express adesired protein (e.g., a CAR). The infused cell is able to kill tumorcells in the recipient.

The present invention includes a method of enhancing the immune responseduring adoptive T cell transfer comprising the steps of contacting oneor more T cells with an inhibitor of one or more DGK isoform. Themodified T cells of the invention may also serve as a type of vaccinefor ex vivo immunization and/or in vivo therapy in a mammal Preferably,the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding theinhibitor to the cells, and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art. Briefly, cells areisolated from a mammal (preferably a human) and modified to enhance itscytolytic activity according to the methods of the invention. Forexample, the cell is modified to have any one or more DGK isoforminhibited. The heighted immunogenic cell can be administered to amammalian recipient to provide a therapeutic benefit. The mammalianrecipient may be a human and the cell so modified can be autologous withrespect to the recipient. Alternatively, the cells can be allogeneic,syngeneic or xenogeneic with respect to the recipient.

In addition to using a cell-based therapy in terms of ex vivomodification of T cells, the present invention also providescompositions and methods for in vivo therapies to elicit an immuneresponse directed against an antigen in a patient.

In one embodiment, the mammal has a type of cancer which expresses atumor-specific antigen. In accordance with the present invention, acomposition can be made which recognizes a specific tumor-specificantigen. In such cases, the inhibitor of one or more DGK isoform isadministered to a T cell that is directed to a given tumor-specificantigen, resulting in an improved therapeutic outcome for the patient,evidenced by, e.g., a slowing or diminution of the growth of cancercells or a solid tumor which expresses the tumor-specific antigen, or areduction in the total number of cancer cells or total tumor burden.

In another embodiment, the compounds of the present invention may beused in combination with existing therapeutic agents used to treatcancer. In some instances, the compounds of the invention may be used incombination these therapeutic agents to enhance the antitumor effect ofthe therapeutic agent.

In order to evaluate potential therapeutic efficacy of the compounds ofthe invention in combination with the antitumor therapeutics describedelsewhere herein, these combinations may be tested for antitumoractivity according to methods known in the art.

In one aspect, the present invention contemplates that the inhibitors ofthe invention may be used in combination with a therapeutic agent suchas an anti-tumor agent including but not limited to a chemotherapeuticagent, an anti-cell proliferation agent or any combination thereof.

The invention should not be limited to any particular chemotherapeuticagent. Rather, any chemotherapeutic agent can be linked to theantibodies of the invention. For example, any conventionalchemotherapeutic agents of the following non-limiting exemplary classesare included in the invention: alkylating agents; nitrosoureas;antimetabolites; antitumor antibiotics; plant alkyloids; taxanes;hormonal agents; and miscellaneous agents.

Alkylating agents are so named because of their ability to add alkylgroups to many electronegative groups under conditions present in cells,thereby interfering with DNA replication to prevent cancer cells fromreproducing. Most alkylating agents are cell cycle non-specific. Inspecific aspects, they stop tumor growth by cross-linking guanine basesin DNA double-helix strands. Non-limiting examples include busulfan,carboplatin, chlorambucil, cisplatin, cyclophosphamide, dacarbazine,ifosfamide, mechlorethamine hydrochloride, melphalan, procarbazine,thiotepa, and uracil mustard.

Anti-metabolites prevent incorporation of bases into DNA during thesynthesis (S) phase of the cell cycle, prohibiting normal developmentand division. Non-limiting examples of antimetabolites include drugssuch as 5-fluorouracil, 6-mercaptopurine, capecitabine, cytosinearabinoside, floxuridine, fludarabine, gemcitabine, methotrexate, andthioguanine.

There are a variety of antitumor antibiotics that generally prevent celldivision by interfering with enzymes needed for cell division or byaltering the membranes that surround cells. Included in this class arethe anthracyclines, such as doxorubicin, which act to prevent celldivision by disrupting the structure of the DNA and terminate itsfunction. These agents are cell cycle non-specific. Non-limitingexamples of antitumor antibiotics include dactinomycin, daunorubicin,doxorubicin, idarubicin, mitomycin-C, and mitoxantrone.

Plant alkaloids inhibit or stop mitosis or inhibit enzymes that preventcells from making proteins needed for cell growth. Frequently used plantalkaloids include vinblastine, vincristine, vindesine, and vinorelbine.However, the invention should not be construed as being limited solelyto these plant alkaloids.

The taxanes affect cell structures called microtubules that areimportant in cellular functions. In normal cell growth, microtubules areformed when a cell starts dividing, but once the cell stops dividing,the microtubules are disassembled or destroyed. Taxanes prohibit themicrotubules from breaking down such that the cancer cells become soclogged with microtubules that they cannot grow and divide. Non-limitingexemplary taxanes include paclitaxel and docetaxel.

Hormonal agents and hormone-like drugs are utilized for certain types ofcancer, including, for example, leukemia, lymphoma, and multiplemyeloma. They are often employed with other types of chemotherapy drugsto enhance their effectiveness. Sex hormones are used to alter theaction or production of female or male hormones and are used to slow thegrowth of breast, prostate, and endometrial cancers. Inhibiting theproduction (aromatase inhibitors) or action (tamoxifen) of thesehormones can often be used as an adjunct to therapy. Some other tumorsare also hormone dependent. Tamoxifen is a non-limiting example of ahormonal agent that interferes with the activity of estrogen, whichpromotes the growth of breast cancer cells.

Miscellaneous agents include chemotherapeutics such as bleomycin,hydroxyurea, L-asparaginase, and procarbazine that are also useful inthe invention.

An anti-cell proliferation agent can further be defined as anapoptosis-inducing agent or a cytotoxic agent. The apoptosis-inducingagent may be a granzyme, a Bcl-2 family member, cytochrome C, a caspase,or a combination thereof. Exemplary granzymes include granzyme A,granzyme B, granzyme C, granzyme D, granzyme E, granzyme F, granzyme G,granzyme H, granzyme I, granzyme J, granzyme K, granzyme L, granzyme M,granzyme N, or a combination thereof. In other specific aspects, theBcl-2 family member is, for example, Bax, Bak, Bcl-Xs, Bad, Bid, Bik,Hrk, Bok, or a combination thereof.

In additional aspects, the caspase is caspase-1, caspase-2, caspase-3,caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9,caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, or acombination thereof. In specific aspects, the cytotoxic agent is TNF-α,gelonin, Prodigiosin, a ribosome-inhibiting protein (RIP), Pseudomonasexotoxin, Clostridium difficile Toxin B, Helicobacter pylori VacA,Yersinia enterocolitica YopT, Violacein, diethylenetriaminepentaaceticacid, irofulven, Diptheria Toxin, mitogillin, ricin, botulinum toxin,cholera toxin, saporin 6, or a combination thereof.

In some embodiments, an effective amount of a compound of the inventionand a therapeutic agent is a synergistic amount. As used herein, a“synergistic combination” or a “synergistic amount” of a compound of theinvention and a therapeutic agent is a combination or amount that ismore effective in the therapeutic or prophylactic treatment of a diseasethan the incremental improvement in treatment outcome that could bepredicted or expected from a merely additive combination of (i) thetherapeutic or prophylactic benefit of the compound of the inventionwhen administered at that same dosage as a monotherapy and (ii) thetherapeutic or prophylactic benefit of the therapeutic agent whenadministered at the same dosage as a monotherapy.

Screening Agents

The samples used in the detection methods of the present inventioninclude, but are not limited to, cells or tissues, protein, membrane, ornucleic acid extracts of the cells or tissues, and biological fluidssuch as blood, serum, and plasma. The sample used in the methods of theinvention will vary based on the assay format, nature of the detectionmethod, and the tissues, cells or extracts which are used as the sample.Methods for preparing protein extracts, membrane extracts or nucleicacid extracts of cells are well known in the art and can be readily beadapted in order to obtain a sample which is compatible with the methodutilized (see, for example, Ausubel et al., Current Protocols inMolecular Biology, Wiley Press, Boston, Mass. (1993)).

Candidate compounds are screened for the ability to inhibit any one ormore DGK isoform. The determination of the inhibitory function of thecandidate agent to any one or more DGK isoform may be done in a numberof ways. In any event, the candidate agent should increase the cytolyticactivity of the cell compare to a cell not contacted with the agent.

The method of identifying an agent capable of inhibiting any one or moreDGK isoform includes the initial step of contacting a cell with theagent and determining the activity or level of any one or more DGKisoform. A decrease in the activity or level of any one or more DGKisoform indicates that the agent is an inhibitor. Preferably, the agentis also capable of enhancing the cytolytic activity of a cell.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1 Augmenting Efficacy of Chimeric Antigen Receptor T cells

The loss of anti-tumor efficacy of CD8 T cells within murine tumors hasbeen recognized for many years. However, virtually nothing is knownabout the effects of the tumor microenvironment on genetically alteredCAR-T cells. Described herein is the identification of a new targetwithin T cells, the molecule Diacylgylceral Kinase (DGK), which appearsto limit T cell killing and cytokine release. By genetically inhibitingthis pathway in CAR T cells, their anti-tumor efficacy is able to beincreased. The results presented herein demonstrate that inhibition ofDGK can augment adoptive T cell transfer.

Activation of T cells through the T-cell antigen receptor (TCR) orthrough CAR, results in a series of molecular events that culminate in Tcell activation. Perhaps the most crucial event downstream of thesereceptors is in the cleavage of the phospholipid PIP2(phosphotidyl-4,5-inositol) by phospholipase C g1 (PLCg1) into thesecond messengers diacylglycerol (DAG) and inositol-1,4,5-phosphate(IP3). Whereas IP3 serves to activate calcium signaling in T cells, DAGbinds and activates proteins important in T cell activation, such as theRas activating protein RasGRP1. At the same time that T cells produceDAG, DGK's are recruited to sites of T cell signaling to phosphorylateDAG to form phosphatidic acid, terminating DAG-mediated signaling (Zhenget al., 2008, EMBO Rep. 9:50-55; Zhong et al., 2008, Immunol. Rev.224:249-264). There are a number of DGK isoforms, but DGKα and DGKζappear to be the most important (Zhong et al., 2008, Immunol. Rev.224:249-264). Deletion of DGK thus results in T cells that are lesscapable of “turning-off” DAG-mediated signaling within T cells. It hasbeen previously shown that deletion of either isoform of DGK, DGKα orDGKζ, results in T cells that demonstrate enhanced signal transductionand appear more resistant to anergy-inducing stimuli, but that do notappear to induce spontaneous auto-immunity. Recent work by Prinz et al.(Prinz et al., 2012, J. Immunol. 188:5990-6000) in freshly isolatedhuman tumor infiltrating lymphocytes (TIL) from renal cell cancersrevealed elevated levels of DGKα in their hypofunctional TIL. They foundthat pharmacologic inhibition of DGK with DGK Type 1 inhibitor couldimprove TIL function. Thus, it was examined whether deletion of eitheror both isoforms of DGK could result in more potent responses downstreamof CAR signal transduction (Zhong et al., 2003, Nat. Immunol.: 882-890;Olenchock et al., 2006, Nat. Immunol. 7:1174-1181; Zha et al., 2006,Nat. Immunol. 7:1166-1173).

The results presented herein also show that intravenous injection of theDGK KO mesoCAR T cells augments efficacy. This was important as thiswould be the preferred route of administration in human patients.

The materials and methods employed in these experiments are nowdescribed.

Chimeric Antigen Receptor (CAR)

A CAR (FIG. 1) is generally comprised of three main components: 1) theexternal single chain fragment variable region (scFv) derived from thelinked V_(H) and V_(L) domains of the antigen binding region of amonoclonal antibody (mAb), 2) a flexible hinge sequence, such as from aCD8α or immunoglobulin sequence, 3), and one or more intracellularsignaling domains, which may include cytoplasmic domains from CD3-ε,CD3-γ, or CD3-ζ from the TCR construct or high-affinity receptor FcεRI.T cells expressing the “first generation” CARs, which signaled solelyvia a lone CD3-ζ domain, were found to be rapidly lost, so thatperipheral T cell persistence was very short in a number of earlyclinical trials (Jensen et al., 2010, Biol. Blood Marrow Transplant16:1245-1256; Till et al., 2008, Blood 112:2261-2271; Pule et al., 2008,Nat. Med. 14:1264-1270; Park et al., 2007, Mol. Ther. 15:825-833).However, the CAR design has evolved to contain up to 3 internal signaldomains (e.g. chimeric CD28, CD134, CD137 (41BB), Lck, ICOS, and DAP10that enhance T cell activation and survival upon antigen binding (Zhaoet al., 2009, J. Immunol. 183:5563-5574; Kowolik et al., 2006, CancerRes. 66:10995-11004; Wang et al., 2007, Hum. Gene Ther. 18:712-725; Yvonet al., 2009, Clin. Cancer Res. 15:5852-5860; Milone et al., 2009, Mol.Ther. 17:1453-1464). The “right” combination of internal signalingdomains needs to induce CAR-dependent killing, but avoidsupraphysiologic T cell activation which may lead to T-cell death orsystemic toxic effects (Morgan et al., 2010, Mol. Ther. 18:843-851). Themain advantages of CAR technology include: i) harnessing the highaffinity/specificity antigen binding of the scFv, ii) freedom from MHCrestriction, and iii) the relative ease with which patient-derived bloodlymphocytes can be expanded and transduced with the desired CAR (June,2007, J. Clin. Invest. 117:1466-1476; Jena et al., 2010, Blood116:1035-1044; Peinert et al., 2009, Immunotherapy 1:905-912; Eshhar,2010, Curr. Opin. Mol. Ther. 12:55-63). Despite extensive research aboutsignaling through the native T cell receptor (TCR), there has beenrelatively little research done on CAR signaling, especially in humanCAR T cells. Although the “basics” seem to be similar between TCR andCAR signaling, there are likely some differences, since unlike signalingthrough a standard TCR, CAR signaling will not involve the CD8 molecule,nor the majority of the CD3 complex.

Mouse Models

The techniques necessary to transduce activated mouse T cells with CARsusing modified retroviruses were developed. After genetic engineering,between 40-70% of the T cells expressed a CAR targeting the humanprotein mesothelin on the surface of mouse or human tumor cells (FIG.2). When added to mesothelin-expressing tumor cells, the mouse T cellCARs can kill about 20% of tumor cells over 24 hours at a ratio of 40 Tcells per one tumor cell (FIG. 3).

It was examined whether mouse T cells lacking DGKζ, DGKα or both, mightpersist longer in mice and retain greater activation. Accordingly, MouseT cells from wild type mice (WT), DGKα KO mice, and DGKζ KO mice weretransduced with the mesothelin CAR transcript and were observed toachieved the same levels of expression (˜60%). As a prelude to studyingtheir effect after IV injection into tumor-bearing mice, their in vitroability to kill tumor cells and secrete the inhibitory cytokineinterferon-γ was examined Transduced T cells were mixed at varyingratios with tumor cells expressing mesothelin (AE17meso) or with tumorcells which did not express mesothelin (AE17). The cells were allowed toco-incubate for 18 hours. At that time, the percent killing of tumorcells was determined and supernatants were removed for measurement ofINFγ.

The results of the experiments are now described.

Loss of DGKζ, DGKα or Both Markedly Enhanced the Ability of the T Cellsto Kill Tumor Cells

Unexpectedly, loss of DGKζ, DGKα or both markedly enhanced the abilityof the T cells to kill tumor cells (FIGS. 4 and 5) and substantiallyincreased the amount of IFNγ released (FIG. 6). Thus, at a ratio of 40 Tcells to one tumor cell, the standard CAR T cells killed about 15-20% ofthe tumors. This killing rate was nearly triple (90-95%) in the DGKα orDGKζ KO T cells. The CAR T cells in the double knockout (DKO) cells(lacking both isoforms of DGK) were able to kill 90% of the tumor cells.The DKO cells had activity at a very low E:T ratio (1:5), while killingwas not seen at this ratio with the control CAR T cells. A similarsynergy was seen in IFNγ release. FIG. 7 shows that when incubated at a20:1 ratio for 20 hours, T cells from DGK knockout mice significantlyincreased killing compared to wild type. Furthermore, the AE17 cellswere not killed (FIG. 7).

It was also observed that elimination of DGK made T-cells less sensitiveto TGFβ-mediated-inhibition of tumor killing. As shown in FIG. 8A, whenWT-mesoCAR T cells are exposed to TGFβ, their ability to kill tumor cellis decreased by more than 60%. However, TGFβ had virtually no effect onthe ability of DGKα KO cells to kill tumor cells. It was also observedthat TGFβ decreased the amount of IFNγ that was released (FIG. 8B). Thiswas also blunted in the DGKα cells. These effects are likely to beimportant in a therapeutic context since tumors make large amounts ofTGFβ.

It was also observed that elimination of DGK made T-cells less sensitiveto PGE2 and adenosine-mediated inhibition of tumor killing. As shown inFIG. 9, when WT-mesoCAR T cells are exposed to PGE2, their ability tokill tumor cell was almost completely inhibited. However, PGE2 inhibitedthe ability of DGKα KO or DGKz KO cells to kill tumor cells by onlyabout 30%. Very similar results are shown in FIG. 10, where the abilityof adenosine to inhibit tumor cell killing is markedly blocked in the KOcells. These effects are likely to be important in a therapeutic contextsince tumors make large amounts of adenosine and PGE2 which inhibit Tcell function.

To determine the impact of DGK loss in an in vivo context, equal numbersof each type of wild type and DGK knockout T cells were mixed withmesothelin-expressing tumor cells, which were then injected into theflanks of B6 mice. Measuring the tumor sizes at 2 weeks provided anestimation of the killing efficacy of the T cells. FIG. 11 shows thatwhile all of the T cells inhibited tumor growth, DGK KO cells were farmore effective. The DKO cells actually cured all of the mice of tumors.Even when a very low ratio of T cells to tumor cells was used (1 T cellto 10 tumor cells) WT CAR inhibited growth by about 24%, while bothtypes of DGK KO T cells inhibited tumor growth by nearly twice as much(43-46%) (FIG. 12).

In another experiment, various types of T cells were injectedintratumorally, and the size of tumors measured after 4 days. As shownin FIG. 13, the DGKα knockout and DKO cells were more effective inreducing tumor size.

In addition, experiments were conducted to mimic potential human trialsin which mice with established tumors were injected intravenously with10⁷ wild type T cells or the three types of DGK knockout T cells. Asshown in FIG. 14, whereas the wild type meso-CAR T cells had no realantitumor effect, under these conditions, all three types of KO T cellsreduced the tumor size by about 50%, showing they have much moreanti-tumor activity than WT CAR T cells. Preliminary relevantobservations in human CART cells have been recorded using a commerciallyavailable DGK inhibitor (R59022). Human mesothelin-CAR T cells mixedwith human mesothelin tumor cells (at a 1:1 ratio) were injected intoimmunodeficient mice. The tumor growth was slowed, but not prevented bythe CAR-T cells. After 2-3 weeks, tumors were harvested and the humanCAR-T cells were purified. When these T cells (Tumor infiltratinglymphocytes, or TILs) are allowed to react with fresh tumors, they cankill only 5-10% of the tumor cells showing they are hypofunctionalcompared to freshly made CAR-T cells which kill 70% of tumor cells (FIG.15). Although this inhibitor is not entirely specific, and not wishingto be bound by any particular theory, these data support the idea thatinhibiting DGK in human CAR-TIL augments function. As described above,these data are very similar to that seen in human TIL (Prinz et al.,2012, J. Immunol. 188:5990-5600).

In another study, a human codon-optimized version of a dominant negative(DN) DGKα with insertion of a myc tag and the proper restriction sitescan be placed into a bicistronic lentiviral vector with a CAR, and usedto transfect human T cells. The presence of the DN construct can beconfirmed using the myc-tag. Effective knockdown of DGK can be confirmedby showing increased active ras production and presumably increasedphospho-ERK. After in vitro assays, the signaling, cytokine release, andtumor killing ability of the CAR-DGK DN T cells can be tested andcompared to CAR T cells.

The CAR-DGK DN T cell TILs show enhanced signaling and cytokine releasewhen exposed to antigen coated beads and enhanced antigen specific tumorkilling. Enhanced anti-tumor activity can be seen in the animal models.The CAR-TILs isolated can be less hypofunctional with improved cytokinerelease and killing when exposed to fresh tumor cells.

A siRNA/shRNA approach can also be undertaken. Validated siRNAs andshRNAs for DGKα are commercially available from Origene, Santa Cruz, andother companies. These can be tested in the CAR-T cells using anti-DGKαstaining and the most effective CAR-T cells subcloned into thelentiviral vector with the H1 promoter (An et al., 2006, Mol. Ther.14:494-504). Zinc finger nucleases can also be used to inhibit DGKαactivity.

As described herein, it was observed that that loss or blockade of DGKisoforms augments efficacy of adoptive T cell transfer. DGK inhibitorsor genetic reduction of DGK (using, for example, shRNA or zinc-fingernucleases) can be used to augment the efficacy of adoptive transfer ofhuman T cells (containing CARs or transgenic T cell receptors) byincreasing their effector function. This would include, but is notlimited to, blood T cells, cord blood T cells, bone marrow T cells, andT cells derived from iPSC. This technology may be used to enhanceefficacy of T cells for use in tumors and to enhance response in chronicinfections such as Hepatitis C virus and HIV virus.

Example 2 Enhanced Effector Responses in Activated CD8+ T CellsDeficient in Diacylglycerol Kinases

Recent clinical trials have shown promise in the use of chimeric antigenreceptor (CAR)-transduced T cells; however, augmentation of theiractivity may broaden their clinical use and improve their efficacy.Since CAR action requires proteins essential for T-cell receptor (TCR)signal transduction, it was examined herein whether deletion of negativeregulators of these signaling pathways would enhance CAR signaling andeffector T-cell function.

For the studies presented herein, it was chosen to target an inhibitorof diacylglycerol (DAG), an essential second messenger that is createdby the cleavage of phosphatidyl (4,5) inositol bisphosphate byphospholipase Cγ1 (PLCγ1) after PLCγ1 is phosphorylated and activated bythe protein tyrosine kinases that are recruited to the stimulated TCR(Smith-Garvin et al., 2009, Annu Rev Immunol 27:591-619). DAG activatessignaling molecules leading to several second messenger cascades, mostnotably the Ras/ERK pathway that is known to be essential for T-cellactivation (Dower et al., 2000, Nat Immunol 1:317-21). After itsgeneration, DAG is actively metabolized into phosphatidic acid by one ofthe 2 isoforms of diacylglycerol kinases (DGK) present within T cells,DGKα or DGKζ (Topham & Prescott, 2001, J Cell Biol 152:1135-43).Previously, it has been observed that deletion of either DGK isoformpotentiates DAG-mediated Ras and extracellular signal-regulated kinase(ERK) activation and augments TCR-induced cytokine production and T-cellproliferation (Zhong et al., 2003, Nat Immunol 4:882-90; Zha et al.,2006, Nat Immunol 7:1166-73; Olenchock et al., 2006, Nat Immunol7:1174-81). Further, it has been found that deletion of DGKζ results inimproved CD8⁺ T-cell responses by augmenting signaling via the TCR whenmice are challenged with a transplantable subcutaneous tumor (Riese etal., 2011, J Biol Chem 286:5254-65). However, neither the absence ofDGKα or DGKζ is sufficient to enable a completely successful antitumorresponse. It is examined here whether combining CAR therapy with DGKdeficiency might boost the ability of T cells to respond to a tumorchallenge. The data presented herein demonstrates this augmentation,suggesting that such a combined therapeutic approach may have use infuture clinical trials.

The materials and methods employed in these experiments are nowdescribed.

Mice

Mice deficient in dgkα, dgkζ, or both backcrossed to C57Bl/6 have beendescribed previously (Zhong et al., 2003, Nat Immunol 4:882-90;Olenchock et al., 2006, Nat Immunol 7:1174-81). C57Bl/6 mice containinga transgene for the OVAp TCR (OT-I mice) were obtained from the JacksonLaboratories. DGKζ-deficient CD45.2 CD90.2 OT-I mice were created bybackcrossing these 2 strains. All experiments were carried out in mice 6to 12 weeks old.

Listeria Infection and EL4-Ova Tumor Model Experiment

Splenic CD8⁺ T cells were isolated from wild-type or DGKζ-deficientCD45.2 CD90.2 OT-I mice by flow cytometry)(CD8⁺CD44^(lo)) as described(Riese et al., 2011, J Biol Chem 286:5254-65). Twenty thousand cellswere transferred intravenously into CD45.2, CD90.1 recipient micesubsequently infected intravenously with 5,000 cfu Listeria-ova 24 hoursafter T-cell transfer. One week later, CD45.2⁺ donor cells were isolatedfrom spleens of recipient mice according to the manufacturer'sinstructions (Miltenyi Biotec), and 1.5×10⁶ of isolated cells weretransferred intravenously into CD45.1, CD90.2 mice that had beeninoculated with 2.5×10⁵ EL4-ovalbumin (EL4-ova) tumor cells, a murinelymphoma line that stably expresses ovalbumin (Moore et al., 1988, Cell54:777-85), in the right flank 2 weeks prior. Tumors were barelypalpable at time of T-cell transfer. One week later, mice wereeuthanized, tumor size was measured, and T cells from spleens and tumorswere analyzed.

T-Cell Transduction

MesoCAR, a fusion protein that contains the antigen-binding region of anantibody specific for the human tumor antigen mesothelin fused with CD8atransmembrane domain, CD3ζ, and the costimulatory domain of 4-1BB, hasbeen described previously (Moon et al., 2011, Clin Cancer Res17:4719-30). cDNA encoding mesoCAR was subcloned into the MIGRretrovirus (Pear et al., 1993, Proc Natl Acad Sci USA 90:8392-6), whichalso expresses GFP using an internal ribosomal entry site. The sequenceof antimesothelin Fv was provided (Chowdhury & Pastan, 1999, NatBiotechnol 17:568-72). Infective particles were generated from thesupernatants of 293T cells transfected with retroviral vector plasmidand helper plasmids using Lipofectamine 2000 (Invitrogen), as previouslydescribed (Jordan et al., 2006, J Immunol 176:2430-8). Primary murine Tcells were isolated as suggested by the manufacturer (Miltenyi Biotec)from the spleens of wild-type or DGK-deficient mice and incubated in24-well plates [4×10⁶ cells/well in 2 mL T-cell media with 100 U/mLinterleukin (IL)-2] coated with α-CD3 (1 μg/mL) and α-CD28 (2 μg/mL).After 48 hours, cells (1×10⁶ cells/well) were mixed with retrovirus (1mL crude viral supernatant) in a 24-well plate coated with Retronectin(50 μg/mL; Clontech) and centrifuged without braking at room temperaturefor 30 minutes at 1,200 g. After overnight incubation, cells wereexpanded with 50 U/mL of IL-2 for 48 hours.

Coating Beads with Recombinant Human Mesothelin

Target antigens were chemically crosslinked to tosylactivated 4.5 μmDynabeads (Invitrogen, #140-13), using the manufacturers' instructions.In brief, 4×10⁷ beads were incubated 16 to 18 hours at 37° C. in thepresence of 20 mg of recombinant human mesothelin (RayBiotech,#230-00043) in 0.1 mol/L sodium phosphate buffer (pH 7.4) with shaking.After incubation, beads were washed and resuspended in PBS containing0.5% bovine serum albumin to a final volume of 1 mL.

Evaluation of CAR T-Cell Effector Functions

Cytotoxicity and IFN ELISA

A stable cell line of the mouse mesothelioma line AE17 expressing humanmesothelin subsequently engineered to express luciferase has beendescribed (Moon et al., 2011, Clin Cancer Res 17:4719-30; Tchou, et al.,2012, Breast Cancer Res Treat 133:799-804). Cytokine release assays wereconducted by co-culture of T cells with target cells at the describedratios, in triplicate, in 96-well round bottom plates in 200 μL. After18 hours, cell lysis was determined from the detection of luciferasefrom the remaining cells using a previously described assay (Moon etal., 2011, Clin Cancer Res 17:4719-30). An ELISA Kit (Biolegend) wasused to measure IFN-γ.

WINN Assay

A total of 1×10⁶ mesothelin-expressing TC1 cells, a murine non-smallcell lung cancer line with well-established use in the WINN assay(DeLong et al., 2003, Cancer Res 63:7845-52), were coinjected into theright flank along with 2×10⁵ CAR-transduced T cells (routinely 50% ofwhich were gfp positive, and thus transduced with CAR). Ten days later,mice were euthanized, and tumor volume was assessed.

Intravenous Transfer of CAR-T Cells in Mice with Preexisting Tumor

C57Bl/6 mice were inoculated subcutaneously with 2×10⁶ AE17 meso cells.Seven days later, at which point tumors were approximately 100 mm³, micewere injected with 1×10⁷ CAR-transduced T cells intravenously by tailvein. Tumor development was monitored by caliper measurement of tumordiameter over an additional 10 days. Each volumetric determination wasderived from the formula 0.52a²b, with a representing the minor axis andb representing the major axis. Alternately, mice were sacrificed at 3 or6 days after transfer, and the presence of T cells within spleen ortumor was determined by evaluating for gfp expression within T-cellsubsets by flow cytometry.

Expression of Cytotoxic Markers Following CAR Activation.

A total of 2×10⁶ mesoCAR-T cells derived from mouse splenocytes repleteor deficient in DGKs were placed in individual wells of a 24-well platewith or without 2×10⁶ mesothelin-coated beads for 18 hours, at 37° C. inthe presence of 30 U/mL of IL-2. After incubation, T cells were stainedfor the presence of the surface markers TRAIL (eBioscence, #12-5951-82)or FasL (eBioscience, #12-5911-81), or the intracellular markersgranzyme B (BD, #51-2090KZ) or perforin (eBioscience, #12-9392-82),using protocols described by the manufacturer. Flow cytometry histogramsof marker expression were evaluated from cells that were positive forgfp (indicating expression of CAR) and CD8, and negative for CD4.

T-Cell Immunoblotting and CD69 Upregulation

To assess for Erk phosphorylation, 1×10⁶ mesoCAR-transduced T cells wereincubated either with mesothelin- or albumin-coated beads in a 1:4 ratio(cells:beads), or with α-CD3ε antibody at 2.5 μg/mL final concentrationfor indicated time points. Lysates were prepared and immunoblotted forphosphorylated Erk, total Erk, or tubulin (antibodies all from CellSignaling) as previously described (Riese et al., 2011, J Biol Chem286:5254-65). Alternately, protein-bead stimulations were allowed toproceed for 5 hours, and then the surface upregulation of CD69 wasdetermined by flow cytometry.

Primary Human CAR-T-Cell Assays

Primary human T cells were obtained and mock infected or transduced withlentivirus expressing mesoCAR as previously described (Moon et al.,2011, Clin Cancer Res 17:4719-30). A total of 5×10⁶ T cells weresubsequently added to 24-well plates that had been seeded with 5×10⁵cells either from the epithelial mesothelioma (EM) human mesotheliomaline or a stable derivative cell line, EM-meso, engineered to expresshigh levels of mesothelin, in a 24-well dish. After 96 hours ofcoincubation (which included the addition of another 3×10⁵ EM or EM-mesocells at the midpoint of coincubation), cells were resuspended, and Tcells were isolated via Lymphoprep density gradient separation(Axis-Shield) as suggested by the manufacturer. Cells were assessed forviability using Trypan blue, and 1×10⁵ live T cells were cocultured with5×10³ EM-meso-luc cells expressing luciferase in 96-well plates in thepresence or absence of the DGK inhibitors DGK1 (R59022) or DGK2 (R59949;Sigma) at 5 μg/mL. After 18 hours, remaining tumor cells were washed andlysed, and luminescence was evaluated. Cell lysate determinations werecorroborated with visual estimate of remaining numbers of tumor cells.

For studies with TGFβ, 1×10⁵ primary human T cells that had been mockinfected or transduced with lentivirus-expressing mesoCAR werecoincubated with 5×10³ EM-meso-luc cells in the presence of indicatedconcentrations of TGF for 18 hours, and cell numbers were determined asdescribed above.

The results of experiments are now described.

DGKζ-Deficient Activated CD8+ T Cells Show Enhanced Response to Tumor

It has been previously shown that naïve DGKζ-deficient CD8⁺ T cellsspecific for ovalbumin (OT-I cells) are better able to control tumorgrowth and undergo activation than naïve wild-type OT-I cells aftertransfer into mice bearing EL4-ova-expressing tumors (Riese et al.,2011, J Biol Chem 286:5254-65). However, in those studies, implantedtumors were not completely eradicated. It was examined herein whetherthe effect of DGKζ deletion would be improved if, instead of naïve Tcells, activated T cells were used, which could potentially confer amore robust antitumor response. In addition, this approach would moreclosely mirror current clinical trials of adoptive CD8⁺ T-cell tumorimmunotherapy that use cells preactivated before transfer. To generateuniform populations of activated cells, naïve OT-I cells sufficient ordeficient in DGKζ were transferred into congenically marked mice, andthe recipient animals were then infected with Listeria engineered toexpress ovalbumin. One week later, antigen-experienced (CD44-high) donorOT-I cells were recovered from spleens and transferred into EL4-ovatumor-bearing mice. Initially, it was noted that expansion of naïveDGKζ-deficient OT-I cells was more robust when compared with naïvewild-type OT-I cells in response to the antigenic challenge withListeria-ova (FIG. 16A), as in other DGKζ-deficient CD8⁺ T cell-modelsof acute infection (Zhong et al., 2003, Nat Immunol 4:882-90; Riese etal., 2011, J Biol Chem 286:5254-65); however, there was no difference inactivation phenotype of the recovered cells as assessed by CD44expression between the 2 different genotypes. After transfer of equalnumbers of wild-type or DGKζ-deficient effector cells into EL4-ovatumor-bearing mice, it was found that although wild-type OT-I cellsconferred no appreciable antitumor effect, tumors in mice treated withDGKζ-deficient activated OT-I cells were significantly (P=0.05) reducedin size compared with untreated animals (FIG. 16B). DGKζ-deficienteffector cells also persisted in increased numbers within the spleen ofhost animals (FIG. 16C) and were observed in larger quantities withinthe tumors of host animals (FIG. 16D). These data show that deficiencyof DGKζ confers enhanced antitumor potential in preactivated T cells. Asin previous studies with naïve DGKζ-deficient OT-I CD8⁺ T cells,however, it was found that transfer of activated DGKζ-deficient CD8⁺ Tcells was insufficient to completely eradicate tumors, suggesting thatthe strategy of targeting DGKζ alone is insufficient in curtailing theprogression of established tumors.

Deletion of DGKζ Enhances Functional Responses of T Cells Downstream ofCARs

Given that deletion of DGKζ conferred enhanced activity of CD8⁺ T cellsagainst established tumors but did not seem to be curative, it wasexamined whether inhibition of DGK function might augment otherapproaches shown to have efficacy in enhancing T-cell responses againsttumor. Therefore, studies were designed to test the impact of DGKdeficiency on effector function of CAR-expressing T cells. Experimentsfirst tested whether DGKζ loss would augment functional responses afterligation of CARs, similar to the augmentation of TCR-induced functionsthat have been shown previously (Zhong et al., 2003, Nat Immunol4:882-90). For this analysis, mesoCAR was utilized, which is a fusionprotein that has high affinity for the human tumor antigen mesothelin,present on human mesothelioma, pancreatic, and ovarian cancer, coupledto the signaling motifs of the TCR CD3ζ chain and the inducible T-cellcostimulatory receptor 4-1BB. Wild-type or DGKζ-deficient activated OT-Icells were transduced with mesoCAR-expressing retrovirus, resulting inapproximately 50% transduction efficiency (FIG. 17A). Transduction didnot affect the activation state of the T cells, as assessed byexpression of CD44, or expression of the endogenous TCR, as assessedwith tetramer specific for OT-I (FIG. 17A). MesoCAR-transducedDGKζ-deficient and wild-type OT-I cells were then compared in theirability to produce IFNγ and mediate target cell lysis after incubationwith AE17ova-meso, a murine cell line engineered to express bothovalbumin and human mesothelin. Transduced OT-I cells lacking DGKζdisplayed enhanced IFNγ production and enhanced cytotoxicity afterincubation with mesothelioma cell lines (FIG. 17B and FIG. 17C),indicating that deletion of DGKζ enhances the function of CAR-transducedCD8⁺ T cells against AE17 cells that express both ova and mesothelin.

Combined Deletion of DGKζ and DGKα Markedly Enhances T-Cell Responsesafter Stimulation of mesoCAR

The finding that deletion of DGKζ enhanced mesoCAR T-cell functionalresponses suggested that these 2 strategies may be used together forpotentiating CD8⁺ T-cell tumor responses. However, these initialexperiments were complicated by the fact that the target cells expressedantigens for both TCR (ovalbumin) and CAR (mesothelin). Thus, to avoidpotentially confounding results with ovalbumin-specific TCRs,experiments were conducted in non-TCR transgenic animals. Further, DGKαand DGKζ-deficient mice were intercrossed to generate animals deficientin both DGK isoforms to study CAR-T cells generated from double knockout(DKO) mice. Naïve T cells were isolated from wild-type, dgkα−/−,dgkζ−/−, or DKO mice and infected with retrovirus encoding mesoCAR underhigh IL-2 concentrations that favored CD8⁺ T-cell growth (cells were 85%CD8⁺ T cells at the end of incubation). As observed with deletion ofDGKζ in OT-I cells, deletion of either DGKα or DGKζ in this populationof cells expressing the mesoCAR receptor conferred enhanced cytokineproduction and cytotoxicity when the T cells were incubated with tumorcells expressing mesothelin (FIG. 18A and FIG. 18B). Strikingly, DKOcells showed profoundly enhanced effector functions compared with cellswith deletion of either DGK isoform alone or wild-type cells. Theenhanced cytotoxicity observed in these cell lines was mesothelinspecific because mesoCAR-transduced DKO T cells did not lyse cells AE17cells expressing an unrelated antigen (AE17ova cells; FIG. 18C).

It was next evaluated whether the changes in signal transduction thathave been previously observed downstream of the TCR in DGK-deficient Tcells, for example, enhanced Ras/Erk/AP-1 signaling (Zhong et al., 2003,Nat Immunol 4:882-90; Olenchock et al., 2006, Nat Immunol 7:1174-81),were also present downstream of CAR. To that end, a means to stimulatemesoCAR T cells that did not require mesothelin-expressing cells wasdeveloped, because these cells express their own Ras signalingmolecules, such as Erk, that could interfere with identifying changesspecific to T cells after stimulation. For these studies, tosylactivatedbeads coated with albumin (as a control) or beads coated with mesothelinwere used to stimulate the mesoCAR-expressing cells. Althoughphosphorylation of Erk was not observed during incubation with controlbeads, Erk phosphorylation could be readily detected during incubationwith mesothelin-coated beads or, as expected, after stimulation of theTCR complex through CD3ε (FIG. 19A). Moreover, activation of Erk bymesothelin beads required expression of mesoCAR because activation of Tcells transduced with control retrovirus (MIGR) was not observed (FIG.19A, right lanes). To test whether deletion of DGKs enhanced Erkactivation downstream of mesoCAR, this experiment was then repeated withmesoCAR-transduced T cells derived from DKO mice. Similar to theenhanced activation of Erk known downstream of the TCR in T cellsdeficient in DGKs, loss of DGKs augmented the activation of Erkdownstream of mesoCAR (FIG. 19B). The analysis was extended byinvestigating the upregulation of CD69 in mesothelin-stimulatedwild-type or DKO T cells, as CD69 expression is controlled by activationof the transcription factor AP-1 following Ras/Erk signaling (Zhong etal., 2003, Nat Immunol 4:882-90). Consistent with the biochemicalenhancement of Erk activation observed in DKO-transduced T cells,upregulation of CD69 was also increased in mesoCAR DKO T cells comparedwith wild-type cells (FIG. 19C), confirming a role for DGKs inregulation of this pathway downstream of CAR. Together, these datasuggest that DGK influences CAR signaling in a manner similar to the TCRand that the combination of CAR expression and DGK deletion couldrepresent an effective strategy for augmenting CD8⁺ T-cell antitumorresponses.

Deletion of Both DGKs Enhances Activity of T Cells Downstream of CARsAgainst Tumor In Vivo

It was next sought to determine whether deletion of DGK isoformsconferred enhanced antitumor responses in vivo making use of the WINNassay. For these experiments, mice were injected with a mixture ofTC1meso cells, a murine non-small cell lung cancer line (DeLong et al.,2003, Cancer Res 63:7845-52), along with mesoCAR-expressing wild-type Tcells or mesoCAR T cells lacking DGKα, DGKζ, or both DGK isoforms.Although mice that received wild-type mesoCAR-transduced T cells or Tcells lacking a single isoform of DGK were unable to completely controlthe growth of mesotheliomas, DKO T cells eradicated the mesotheliomas(FIG. 20A), indicating that, as suggested by in vitro studies, targetingDGK generates meaningful enhancement of CD8⁺ T cells against tumor.Pronounced differences were also noted when AE17meso cells were used astarget cells in the WINN assay. Although this experiment offeredproof-of-principle that DKO T cells conferred enhanced in vivo activityagainst mesothelioma, it did not directly assess whether deletion of DGKisoforms would be capable of limiting the growth of established tumors,which is more representative of how CAR-T cells would be usedclinically. To that end, AE17meso cells were injected into the flanks ofmice, and tumors were allowed to develop to approximately 100 mm³ insize before intravenous administration of CAR-T cells. Under theseconditions, although wild-type mesoCAR-transduced T cells wereineffective at limiting tumor growth, mesoCAR-transduced T cellsdeficient in either DGK isoform significantly (P<0.05) decreased therate of tumor growth (FIG. 20B), an effect increased by the deletion ofboth DGK isoforms. In addition to enhanced effector function, thiseffect could, in part, be related to the increased number ofDGK-deficient mesoCAR T cells in tumor-bearing mice because quantitativedifferences in T cells between mesoCAR wild-type and DGKζ T cells wereappreciated 6 days after transfer (FIG. 20C); however, mesoCAR T cellsof any genotype were not detected at day 10 or later timepoints underthe experimental conditions used.

Inhibition of DGKs Confers Enhanced Antitumor Responses to Human T Cells

Although the demonstration of enhanced mesoCAR function in murine Tcells lacking DGK isoforms provides a rationale for targeting DGKs toaugment T-cell responses against tumors, an important next step is toestablish a role for DGKs in CAR-expressing primary human T cells. Overthe course of studies with primary human T cells transduced withmesoCARs, it has been noted that these cells develop reduced functionalresponsiveness after extended coculture with mesothelin-expressing tumorcells. As shown, 96 hours of coincubation of mesoCAR T cells withEM-meso cells, a mesothelioma line engineered to express high levels ofmesothelin, results in significant impairment of mesoCAR T cell lysis oftarget cells upon reculture (FIG. 21A, left). This effect is reminiscentof various models of antigen-induced anergy, as impaired cytotoxicity isnot generated after coculture of mesoCAR T cells with parental EM cellsthat do not express mesothelin (FIG. 21A). Because it has beenpreviously shown that deletion of DGKs mitigates the induction of anergy(Olenchock et al., 2006, Nat Immunol 7:1174-81; Zha et al., 2006, NatImmunol 7:1166-73), it was examined whether inhibition of DGKs mightalso diminish the impaired cytotoxicity observed in the assay. To testthis, mesoCAR T cells were incubated with EM-meso cells for 96 hours,and their ability to lyse target cells in the presence of DGK inhibitorsR59022 (DGK1 inhibitor) or R59949 (DGK2 inhibitor) was then assessed. Itwas observed that the addition of either DGK inhibitor was sufficient toreverse the impaired cytotoxicity present in mesothelin-exposed mesoCART cells (FIG. 21A, center and right), indicating that, similar to thefindings in mice, inhibition of DGK function seems to augment antitumoractivity of primary human T cells expressing CARs. These data alsosuggest that in addition to augmenting TCR (or CAR)-mediated signaling,blockade of DGK enhances T-cell antitumor responses by mitigatingantigen-induced unresponsiveness of the effector cells.

DGK-Deficient T Cells Show Reduced Sensitivity to TGFβ

Following the observation that inhibition of DGKs reverses theantigen-induced inactivation of CAR-expressing T cells, it wasinvestigated whether deletion of DGK might also reduce sensitivity toother inhibitory influences of T cells. One such inhibitor, TGFβ, is ofparticular relevance, because secretion of this cytokine by tumor cellshas been shown to actively inhibit CD8⁺ effector T-cell responsesagainst tumors (Flavell et al., 2010, Nat Rev Immunol 10:554-67).Furthermore, TGFβ is speculated to mediate its effects, in part, bydampening the Ras/Erk signal transduction pathway, which is known to beaffected by DGKs (Nakamura et al., 2012, Cancer Sci 103:26-33; di Bari,et al., 2009, Cancer Immunol Immunother 58:1809-18). Initially, it wasexamined whether TGFβ could lead to inhibition of cytotoxicity of humanmesoCAR T cells incubated with EM-meso cells. The addition of TGFβresulted in diminished cytotoxicity by transduced mesoCAR human T cells(FIG. 6B), at levels of TGFβ similar to that present in culture media ofEM-meso cells (81.31 pg/mL/24 hours/10⁶ cells). Next, it was testedwhether deletion of DGKs could diminish the effects of TGFβ. MurinemesoCAR T cells replete or deficient in DGKs were incubated withAE17meso cells as described previously, and cytotoxicity and IFNγproduction were assessed in the presence or absence of TGFβ. As shownbefore, deletion of either or both DGK isoforms resulted in mesoCAR Tcells with enhanced function when compared with wild-type mesoCAR Tcells (FIG. 22A and FIG. 22B, white bars). Addition of TGFβ at lower (1ng/mL) or higher (10 ng/mL) concentrations of TGFβ were found to reducecytotoxicity and IFNγ secretion in wild-type mesoCAR T cells; however,these functions were not affected in mesoCAR T cells deficient in eitheror both DGKs (FIG. 22A and FIG. 22B). These data suggest that deletionof DGKs confers relative resistance to TGFβ for mesoCAR T cells. Thefinding of relative insensitivity to inhibitory stimuli seems not to besolely restricted to TGFβ because greater functional responses were alsoobserved by DGK-deficient mesoCAR-transduced T cells in the presence ofthe inhibitory stimuli PGE2 and adenosine (FIG. 23), although to alesser extent than that observed with TGFβ. Collectively the datapresented herein suggest that deletion of DGKs augments effectorfunction of CAR-expressing CD8⁺ T cells not only by augmenting signalingthrough the CAR itself but also by reducing sensitivity of the effectorcells to physiologically relevant inhibitory signals.

Increased FasL and TRAIL Expression in DGK-Deficient T Cells

Because TGFβ expression suppresses mediators of CD8⁺ T-cell cytotoxicity(Bedi et al., 2012, Mol Cancer Ther 11:2429-39), such as perforin,granzyme B, FasL, and TRAIL, and because CAR T-cell cytotoxicity ismediated through these effector molecules (Cartellieri et al., 2010, JBiomed Biotechnol 2010:956304), it was examined whether DGK-deficientCAR cells would show greater upregulation of these cytotoxic mediatorswhen compared with wild-type T cells and whether this upregulation maybe the basis for the enhanced cytotoxicity observed in DGK-deficientmesoCAR T cells. Wild-type mesoCAR T cells or mesoCAR T cells deficientin one or both DGK isoforms were exposed to mesothelin-coated beads inthe presence of IL-2 for 18 hours. Expression of FasL, TRAIL, granzymeB, and perforin were then evaluated by flow cytometry. As predicted,mesoCAR T cells deficient in one or both DGKs showed enhanced expressionof the cytotoxic cell surface proteins FasL and TRAIL when compared withwild-type mesoCAR T cells (FIG. 24). In contrast, no difference wasobserved in the expression of the intracellular cytotoxic proteinsgranzyme B- or perforin expressing in cells lacking DGKs (FIG. 25).These data suggest that FasL and TRAIL help facilitate the augmentedcytotoxicity observed in DGK-deficient mesoCAR T cells.

The Role of DGKs in Limiting the Effector Function of Activated CD8+ TCells

Current strategies aimed at augmenting T-cell immune responses againstmalignancy have focused either on assisting the initial activation orpriming of naïve T cells, or on potentiating the effects of activated orprimed T cells. For instance, antibodies that activate CD40 on APCsupregulate costimulatory molecules that help facilitate priming of naïvecells (Ridge et al., 1998, Nature 393:474-8; Schoenberger et al., 1998,Nature 393:480-3; Bennett et al., 1998, Nature 393:478-80). In contrast,antibodies that block the T-cell inhibitory cell surface molecule CTLA-4minimally impact naïve T cells but significantly enhance proliferationand effector function of primed T cells (Chambers et al., 1998, Eur JImmunol 28:3137-43; Gajewski et al., 2001, J Immunol 166:3900-7)Inhibition of proteins that negatively regulate signal transductiondownstream of the TCR has garnered recent attention as a potentialstrategy for augmenting T-cell responses to malignancy at the time of Tcell priming. For instance, deletion of Casitas-B-lineage lymphoma b(cb1-b), an E3 ubiquitin ligase responsible for the degradation ofseveral proteins important in TCR signal transduction, results in Tcells with a decreased requirement of costimulation at the time ofactivation and enhanced antitumor activity of naïve T cells (Chiang etal., 2007, J Clin Invest 117:1029-36; Loeser et al., 2007, J Exp Med204:879-91; Stromnes et al., 2010, J Clin Invest 120:3722-34; Wallner etal., 2012, Clin Dev Immunol 2012:692639).

It has been previously shown that, similar to cb1-b, deletion of DGKζenhanced the effector functions of naïve CD8⁺ T cells (Riese et al.,2011, J Biol Chem 286:5254-65). Although deletion of cb1-b and DGKζ bothresults in changes downstream of the TCR, DGKζ, and DGKα, likely actdirectly to regulate the threshold for activation of T cells downstreamof the TCR. Under a currently posited model of TCR signaling, theinterplay of 2 Ras-activating proteins, the guanine nucleotide-exchangefactors SOS and RasGRP1, determine whether the threshold for Rasactivation is met within a T cell after TCR engagement, an eventrequired for T-cell activation (Das et al., 2009, Cell 136:337-51). Inthis model, TCR ligation results in the production of DAG, which bindsand activates RasGRP1, and generates small amounts of active Ras. Ifenough active Ras is generated, it is able to bind an allostericRas-binding site on SOS, activating SOS and facilitating generation ofmost of the active Ras within activated T cells. In a manner largelyconsistent with this model, it has been previously found that deletionof DGKζ resulted in a decreased threshold of T-cell activation, afinding that correlated with enhanced responses in naïve CD8⁺ T cells.

In the studies presented herein, it was found that deletion of DGKs alsohas profound effects on activated T cells. After uniform activation ofnaïve wild-type or DGKζ-deficient ovalbumin-specific T cells withListeria-ova and then transfer into mice with subcutaneousovalbumin-expressing EL4 lymphoma, it was found that DGKζ-deficient Tcells showed enhanced activity against tumor and increased persistence,both within the spleen and the tumor itself. This finding suggests thatalteration of T-cell threshold plays an important role at multiplestages with the T-cell life cycle and identifies DGKζ as a means tosimultaneously target both naïve and activated populations of effector Tcells.

The role of DGKs in limiting the effector function of activated CD8⁺ Tcells makes DGKs a potential target for CAR-expressing T cells, astrategy gaining traction in the clinical treatment of humanmalignancies. In current clinical trial protocols, human T cells aretransduced with lentivirus-expressing CARs that contain CD3 and CD28 orCD3 and 41BB (CD137), a process that induces T-cell division andactivation upon tumor antigen binding (Kalos et al., 2011, Sci TranslMed 3:95ra73). However, it is now clear that additional strategies willbe necessary to harness the full potential of CAR-T cells, especially inthe treatment of solid malignancies. Although clinical trials in CLLseem promising (Kalos et al., 2011, Sci Transl Med 3:95ra73; Porter etal., 2011, N Engl J Med 365:725-33), earlier works with CAR-transduced Tcells in solid malignancies, such as ovarian cancer (Kershaw et al.,2006, Clin Cancer Res 12(20 Pt 1):6106-15) and renal cell carcinoma(Lamers et al., 2006, J Clin Oncol 24:e20-2), were less encouraging,with an absence of objective tumor response and the lack of T-cellpersistence. In the studies described here, it was evaluated whetherDGKs represent a possible strategy for augmenting CAR-expressing Tcells.

After establishing a retroviral system to efficiently transduce murine Tcells, it was found, as with TCR signaling, that deletion of DGKζaugmented Erk activation, a phosphorylation event that occurs downstreamof DAG formation, after CAR ligation. Deletion of DGKζ was also found toaugment CAR-dependent effector functions because these cells exhibitedenhanced cytokine production and target cell killing relative to theirwild-type counterparts. Deletion of both T-cell isoforms of DGK resultedin even greater enhancement of effector functions of mesoCAR-transducedcells and resulted in control of tumor in vivo in tumor-bearing mice.These results are encouraging for ongoing clinical trials because murinestudies of CAR-transduced T cells have accurately predicted clinicaloutcomes in past trials (Milone et al., 2009, Mol Ther 17:1453-64; Kaloset al., 2011, Sci Transl Med 3:95ra73; Carpenito et al., 2009, Proc NatlAcad Sci USA 106:3360-5).

Although CARs are uniquely positioned to deal with the limited presenceof antigen and costimulation found within the tumor environment, they donot address a third issue relating to T-cell response to tumor:inhibitory stimuli. In these studies, deletion of DGKs was uncovered asa novel strategy for enhancing T-cell activity in the presence ofinhibitory stimuli. Specifically, it was found that prolonged exposureto antigen or the tumor microenvironment inhibitors PGE2, adenosine, andTGFβ were less able to suppress CD8⁺ effector functions in T cells thatlacked one or both T-cell isoforms of DGK. TGFβ is thought to be a keymediator of tumor-mediated inhibition because it is secreted by avariety of tumors, and inhibition of TGFβ signaling, through theexpression of a dominant negative receptor, results in enhancedtumor-specific activity of cytotoxic lymphocytes (Gorelik & Flavell,2001, Nat Med 7:1118-22; Bollard et al., 2002, Blood 99:3179-87). Infact, the amount of TGFβ produced by human cancers, such as prostatecancer, inversely correlates to a patient's overall prognosis (Wikströmet al., 1998, Prostate 37:19-29; Eastham et al., 1995, Lab Invest73:628-35). The enhanced Ras activation imparted by the loss of DGKsmight explain how DGK-deficient lymphocytes develop insensitivity toTGFβ. Because TGFβ is known to result in the reduced phosphorylation ofItk, a Tec kinase important in PLCγ1 activation (Chen et al., 2003, JExp Med 197:1689-99), and because PLCγ1 is the protein directlyresponsible for DAG generation in T cells, one could envision thatdeletion of DGKs might directly subvert this TGFβ-induced signalingalteration.

It has been shown herein that pharmacologic inhibition of DGKs augmentthe efficacy of human CART cells under inhibitory in vitro conditions.This finding suggests that DGKs play an important role in human T cellsand that DGKs may represent attractive clinical targets in augmentingCAR T-cell-based therapies. Model systems in which DGK activity issuppressed through decreased DGK expression (e.g., through expression ofshRNA specific for DGKs) or inhibited DGK function (e.g., throughexpression of dominant negative forms of DGKs) are being developed. Ofcourse, as one develops more potent CART cells, issues of toxicity maybecome relevant. toxicity We could not be assessed in the present modelsystem because the CAR T cells are specific for human mesothelin and donot react with an endogenous mouse protein. However, a second model hasbeen developed using CARs specific for the murine antigen mousefibroblast activation protein (FAP), an antigen overexpressed oncancer-associated fibroblasts. In initial studies, enhanced antitumorefficacy has been observed using DGKζ-deficient FAP-CAR T cells intumor-bearing mice, without evidence of enhanced toxicity. One approachthat is used when introducing CAR T cells with augmented function is tobegin the trial using T cells transduced with short-lived CAR mRNA (Zhaoet al., 2010, Cancer Res 70:9053-61), thus mitigating the potential forchronic CAR-induced autoimmunity.

The data presented herein supports the notion that combining CARexpression, which improves targeting of T cells to tumors and drives aninitial stimulatory response, with inhibition of proteins known to bluntthe effectiveness of the TCR signal may synergize to drive an effectorresponse. The additional value of creating effector T cells resistant tothe inhibitory environment generated by the tumor is also likely tocontribute to the enhanced efficacy observed in this combined approach.Collectively, the data demonstrates that targeting DGKs, as one means toblunt an endogenous inhibitory response, is a useful mechanism toimprove CAR-based strategies in the treatment of human malignancy.

Example 3 Deletion of DGK Enhances Effector Functions and TherapeuticResponse of FAP-CAR T Cells that Target Tumor Stroma

As presented herein, experiments were conducted utilizing a differentCAR targeting a completely different target, fibroblast activationprotein (FAP). It is shown that the efficacy of DGKζ-deficient CAR Tcells is similarly enhanced as the data using the mesothelin CARpresented elsewhere herein. Fibroblast activation protein is proteinexpressed on tumor-associated fibroblasts and could be a good target foranti-cancer therapy.

The materials and methods used in these experiments are now described.

Synthesis of Anti-muFAP CAR Constructs

Total RNA from 73.3 hybridoma cells was isolated and reverse transcribedto cDNA. Variable heavy (V_(H)) and light (V_(L)) chains of 73.3anti-muFAP antibody were PCR amplified and sequenced. The V_(H) andV_(L) sequences were fused with a CAR construct that is being evaluatedin clinical trials (CD8α hinge, CD8α transmembrane domain, and two humanintracellular signaling domains derived from 4-1BB and CD3ζ). This CARwas then inserted into a retroviral MigR1 vector (FIG. 26) that alsoexpresses green fluorescent protein (GFP) for tracking purposes (Pear WS et al, 1998, Blood, 92(10): 3780-3792). Infective particles weregenerated from the supernatants of 293T cells transfected withretroviral vector plasmid and helper plasmids using Lipofectamine 2000(Invitrogen).

Isolation, Transduction and Expansion of Primary Mouse T Lymphocytes

Primary murine splenic T cells from regular C57BL/6 or DGKζ KO mice wereisolated and transduced using the “Pan T cell Negative Selection” kit assuggested by the manufacturer (Miltenyi Biotec), and incubated in24-well plates (4×10⁶ cells/well in 2 mL supplemented RPMI-1640 with 100U/mL IL-2) pre-coated with α-CD3 (1 μg/mL) and α-CD28 (2 μg/mL). After48 hours, cells (1×10⁶ cells/well) were mixed with retrovirus (1 mLcrude viral supernatant) in a 24-well plate coated with Retronectin (50μg/mL; Clontech) and centrifuged, without braking, at room temperaturefor 45 minutes at 1200 g. After overnight incubation, cells wereexpanded with 50 U/mL of IL-2 for additional 48 hours.

Cytotoxicity and IFNγ ELISA

Parental 3T3 and 3T3.FAP cells were transduced with luciferase aspreviously described (Moon et al, 2011, Clin Cancer Res, 17: 4719-4730).Cytotoxicity assays were performed by co-culture of T cells with target3T3 cells at the indicated ratios, in triplicate, in 96-well roundbottom plates. After 18 hours, the culture supernatants were collectedfor IFNγ analysis using an ELISA (mouse IFNγ, BD OpEIA). Cytotoxicity oftransduced T cells was determined by detecting the remaining luciferaseactivity from the cell lysate.

Transfer of CAR-T Cells into Mice Bearing Established Tumors.

Mice were injected subcutaneously with 2×10⁶ AE17.ova (into C57BL/6mice) tumor cells into the dorsal-lateral flank. Tumor-bearing mice (100mm³) were then randomly assigned to remain untreated or to receiveFAP-CAR T cells with or without DGKζ deletion (minimum, five mice pergroup, each experiment repeated at least once). 10⁷ T cells wereadministered through the tail vein. The tumor size were measured bycalipers. At the end of the experiment, tumors and spleens wereharvested for flow cytometric analyses.

Flow Cytometric Analyses

Tumors were harvested at 11 days after adoptive transfer of FAP-CAR Tcells to check for persistence of WT and DGKζ KO FAP-CAR T cells. Cellacquisition was performed on LSR-II using FACSDiva software (BDBioscience, USA). Data were analyzed using FlowJo (Tree Star).

The results of the experiments are now described.

As mesothelin-targeted CAR mouse T cells deficient in the inhibitoryenzyme diacylglycerol kinase zeta (DGKζ) had enhanced effector functionsin vitro and in vivo and increased persistence, the efficacy ofcomparably transduced FAP-CAR splenic T cells isolated from WT C57BL/6versus DGKζ-null mice was evaluated. DGKζ knockout FAP-CAR T cells weremore efficient in lysing 3T3.FAP cells (FIG. 27) and in secreting moreIFNγ (FIG. 28) with retention of specificity in vitro. TheDGKζ-deficient FAP-CAR T cells were also more significantly moreefficient (p<0.05 on day 11) after being injected into AE17.ova bearingmice (FIG. 29). The increased efficacy was associated with greaterpersistence of the DGKζ-knockout compared to WT FAP-CAR T cells (GFP⁺cells) (FIG. 30). Thus, the enhanced anti-tumor efficacy was likely dueto both increased T cell activity and to increased persistence.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed:
 1. A composition for enhancing the cytolytic activityof a cell, said composition comprising an inhibitor of diacylglycerolkinase (DGK) or a downstream effector protein thereof.
 2. Thecomposition of claim 1, wherein the inhibitor is selected from the groupconsisting of a small interfering RNA (siRNA), short hairpin RNA(shRNA), an antisense nucleic acid, a ribozyme, a dominant negativemutant, an antibody, a peptide, a zinc finger nuclease, and a smallmolecule.
 3. The composition of claim 1, wherein the cell is a T cell.4. The composition of claim 3, wherein the T cell is an activated Tcell.
 5. The composition of claim 3, wherein the T cell is modified toexpress a chimeric antigen receptor (CAR).
 6. The composition of claim1, wherein the composition inhibits the DGK isoform selected from thegroup consisting of DGKα and DGKζ.
 7. The composition of claim 1,wherein the composition inhibits both DGKα and DGKζ.
 8. An isolated cellhaving enhanced cytolytic activity, wherein the cell comprises aninhibitor of DGK or a downstream effector protein thereof.
 9. The cellof claim 8, wherein the inhibitor is selected from the group consistingof a small interfering RNA (siRNA), short hairpin RNA (shRNA), anantisense nucleic acid, a ribozyme, a dominant negative mutant, anantibody, a peptide, a zinc finger nuclease, and a small molecule. 10.The cell of claim 8, wherein the cell is a T cell.
 11. The cell of claim10, wherein the T cell is an activated T cell.
 12. The cell of claim 10,wherein the T cell is modified to express a chimeric antigen receptor(CAR).
 13. The cell of claim 8, wherein the composition inhibits the DGKisoform selected from the group consisting of DGKα and DGKζ.
 14. Thecell of claim 8, wherein the cell comprises an inhibitor of DGKα andDGKζ.
 15. A method of enhancing the cytolytic activity of a cell, saidmethod comprising administering to the cell an effective amount of acomposition comprising an inhibitor of DGK or a downstream effectorprotein thereof.
 16. The method of claim 15, wherein the inhibitor isselected from the group consisting of a small interfering RNA (siRNA),short hairpin RNA (shRNA), an antisense nucleic acid, a ribozyme, adominant negative mutant, an antibody, a peptide, a zinc fingernuclease, and a small molecule.
 17. The method of claim 15, wherein thecell is a T cell.
 18. The method of claim 17, wherein the T cell is anactivated T cell.
 19. The method of claim 17, wherein the T cell ismodified to express a chimeric antigen receptor (CAR).
 20. The method ofclaim 15, wherein the composition inhibits the DGK isoform selected fromthe group consisting of DGKα and DGKζ.
 21. The method of claim 15,wherein the composition inhibits both DGKα and DGKζ.
 22. The method ofclaim 15, wherein the cell is genetically modified to express theinhibitor.
 23. The method of claim 15, wherein administering theinhibitor comprises administering the inhibitor in an ex vivoenvironment.
 24. A method of enhancing adoptive T cell transfer in asubject, said method comprising administering to a T cell an effectiveamount of a composition comprising an inhibitor of DGK or a downstreameffector protein thereof, wherein the T cell is administered to thesubject during adoptive T cell transfer.
 25. The method of claim 24,wherein the inhibitor is selected from the group consisting of a smallinterfering RNA (siRNA), short hairpin RNA (shRNA), an antisense nucleicacid, a ribozyme, a dominant negative mutant, an antibody, a peptide, azinc finger nuclease, and a small molecule.
 26. The method claim 24,wherein the T cell is an activated T cell.
 27. The method of claim 24,wherein the T cell is an autologous T cell.
 28. The method of claim 24,wherein the T cell is modified to express a chimeric antigen receptor(CAR).
 29. The method of claim 24, wherein the composition inhibits theDGK isoform selected from the group consisting of DGKα and DGKζ.
 30. Themethod of claim 24, wherein the composition inhibits both DGKα and DGKζ.31. The method of claim 24, wherein the cell is genetically modified toexpress the inhibitor.
 32. The method of claim 24, wherein administeringthe inhibitor comprises administering the inhibitor in an ex vivoenvironment.